{"paper_id":"ee310ac8-b4eb-4df3-990b-e1f8946951ee","body_text":"Glyphosate (GLY), an aminophosphonic\nanalogue of the natural amino\nacid glycine, has been extensively used as an herbicide since the\nearly 1970s due to its ability to inhibit the biosynthesis of essential\naromatic amino acids in plants. The development of genetically modified\n(GM) GLY-resistant crops in 1996 greatly expanded the agricultural\nuse of GLY-based herbicides (GBHs), making them the most widely used\nherbicides worldwide.  In 2024, GBHs were\napplied to over 350 million hectares globally, consuming approximately\n8.6 billion kilograms.  Adoption of GM\nGLY-tolerant crops is responsible for 56% of worldwide GLY consumption.  As a result of its widespread use, GLY and its\nprimary metabolite, aminomethylphosphonic acid (AMPA), are now environmentally\nubiquitous,  and detectable in human urine,\nserum, as well as breast milk. \n , \n  In high-use regions,\nmean urinary concentrations of GLY reached 7.6 μg/L, exceeding\nlevels in the general population (below 4 μg/L).  A 2015–2016 Indiana cohort of 71 pregnant women\nshowed that 93% had detections of urinary GLY (mean: 3.4 μg/L),\nwith rural residents exhibiting elevated concentrations (4.19 μg/L)\nrelative to 3.17–3.47 μg/L in suburban/urban subgroups.  Similarly, in nonfarming households of Iowa,\n88% of children and 65% of mothers had detectable urinary GLY with\nmean concentrations of 2.5 and 1.2 μg/L, respectively.  Given its pervasive environmental presence and\ncontroversial toxicity, risk associated with GLY exposure has become\na critical public health issue.\nThe US Environmental Protection Agency (EPA) has established a\nchronic reference dose (cRfD) for GLY of 1.75 mg/kg/day, while the\nEuropean Union (EU) has set an acceptable daily intake (ADI) of 0.3\nmg/kg/day. In addition, the no-observed-adverse-effect level (NOAEL)\nfor 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\n(GBHs) may act as endocrine disruptors at doses near or below regulatory\nsafety thresholds (e.g., cRfD or ADI), with adverse effects on reproduction. \n , \n  Observed effects include hormonal imbalances, \n − \n \n  proliferation/mitotic\nindex alterations, \n , \n  dysregulation of genes and proteins\ninvolved in endocrine signaling pathways, \n , , \n  induction of oxidative stress,  and epigenetic modifications such as changes\nin DNA methylation levels and histone posttranslational modifications\n(PTMs) in genes associated with endocrine function.  Epidemiological studies have found that GLY exposure levels\nare associated with adverse birth outcomes such as preterm birth and\nbirth defects, as well as alterations in sex hormone levels in adults\nand children. \n , − \n \n  These findings\nhighlight the endocrine-disrupting potential of GLY, primarily affecting\nsex hormones and the female reproductive organs, including the uterus\nand ovary. \n , , \n  Notably, some\nstudies suggest that GLY may induce multigenerational effects, potentially\ntransmitting adverse outcomes across generations. \n ,\nThe toxicity of GLY has been extensively studied in the past\nfew\ndecades. Research on GLY-induced female reproductive toxicity has\nused various  in vivo  and  in vitro  models in toxicology and environmental health studies. Multiple\nmolecular signaling pathways have been implicated in adverse reproduction\neffects in females. However, inconsistencies in findings often arise\nbecause of differences in models and exposure patterns. Given these\ncomplexities, there is an urgent need for a framework that integrates\nmolecular and cellular events with adverse reproductive outcomes in\nindividuals and populations. Such a framework would provide a holistic\nperspective for assessing the toxicity of GLY to the female reproductive\nsystem.\nThe Adverse Outcome Pathway (AOP) framework is a conceptual\nmodel\nthat links molecular initiating events (MIEs) to adverse outcomes\n(AOs) through a series of key events (KEs) connected by key event\nrelationships (KER).  MIEs, which serve\nas the starting point for an AOP, represent specific types of effects\ntriggered by stressors or chemicals that interact with biological\nsystems at the molecular level. KEs are defined as measurable changes\nin cellular or organic function that are linked by KERs and ultimately\nlead to AOs at the organism level.  Since\n2014, 35 AOPs have been officially published in the OECD iLibrary\n( www.oecd-ilibrary.org ), with more than 500 additional AOPs currently under development\non the AOP-wiki ( https://aopwiki.org/aops ). In recent years, AOP frameworks have been increasingly used to\nassess the safety and risk of chemical and environmental exposures,\nenabling the development of risk management strategies based on mechanistic\ntoxicity data. \n , \n  To date, AOPs have not been associated\nwith GLY-induced female reproductive toxicity in the OECD iLibrary,\nAOP-wiki, or published literature.\nThis review integrates  in vivo  and  in\nvitro  evidence to summarize current toxicological knowledge\nand potential mechanisms underlying GLY-induced female reproductive\ntoxicity. Using the AOP-wiki and existing studies, we developed an\nAOP framework to elucidate the causal mechanism of GLY-induced female\nreproductive toxicity at multiple biological levels, from the molecular\nand cellular to the organ, individual, and population levels. This\nframework not only supports early risk assessment of GLY exposure\nbut also identifies potential intervention targets and provides strategies\nto mitigate adverse effects.\n\nA comprehensive literature\nsearch was conducted using the PubMed\nand Web of Science databases, covering articles published in English-language\njournals up to March 2025. The search used keywords such as “glyphosate,”\n“glyphosate-based herbicides,” “female reproduction,”\n“reproductive,” “oocyte,” and “fertility”.\nAll retrieved articles were initially screened using the title and\nabstracts. Studies were excluded if they were: (1) nonoriginal research,\nincluding reviews or commentaries, (2) studies that did not focus\non female reproductive toxicity, and (3) studies that did not use\nmammalian or cell culture models of the reproductive system. The screening\nprocess is shown in  Figure  \n . Following this process, 49 original studies were selected,\nall of which investigated the effects of GLY or GBHs on female reproductive\ntoxicity using mammalian or cell culture models of the female reproductive\nsystem.\nFlow diagram for searching and selecting studies.\nThis review establishes a qualitative AOP framework\nby synthesizing\nthe available evidence in accordance with the development principles\noutlined in the OECD handbook. During the evidence synthesis process,\nwe prioritized mammalian experimental evidence as well as studies\nusing mammalian cell lines and human-derived cell lines, while nonmammalian\nmodel studies were excluded from the material scope of the AOP framework\nconstruction. The graphical linear flow diagram was employed to illustrate\nthe proposed AOP network. The reported end points of GLY or GBH-induced\nfemale reproductive toxicity were classified into MIEs, KEs, and AOs\naccording to the AOP framework. The KEs were further categorized into\nthree biological levels ( Tables  \n  and  S1 ): (1) molecular,\n(2) cellular, and (3) tissue or organic level. Furthermore, we aligned\ninformation on GLY/GBH-induced female reproductive toxicity mechanisms\nwith existing MIEs, KEs, and AOs according to the AOP-wiki. The biological\nplausibility, the essentiality, and empirical support for each KER\nwere evaluated as high, moderate, or low based on the Bradford-Hill\ncriteria and OECD guidelines, \n , \n  and the details of\nthe evaluation criteria are provided in the  Supporting Text 1 . These criteria were used to assess all KERs ( Table  \n ). The following sections\nprovide a comprehensive description of the KEs associated with GLY-induced\nfemale reproductive toxicity at each biological level.\nTo clearly distinguish between hazard identification\nunder high-dose\nexposure and risk characterization under realistic exposure scenarios,\nwe calculated the maximum daily human exposure dose to GLY as 0.22\nμg/kg/day based on the highest reported GLY concentration in\nhuman urine samples (7.6 μg/L) (see  Supporting Text 2  for detailed calculation procedures).  The widely accepted 10-fold safety factor approach was\nemployed to derive human exposure limit standards, incorporating a\nthreshold safety factor when extrapolating experimental data (including\nanimal studies and in vitro test results) to establish a safe reference\ndose for humans. \n , \n  As shown in  Table S1 , all  in vivo  studies are classified\ninto two categories: the first category is human-relevant exposure\ndose studies that can be used for risk characterization, defined as\nexperimental doses ≤10× the estimated maximum daily human\nexposure dose (0.22 μg/kg/day), i.e., ≤2.2 μg/kg/day;\nthe second category is exceed human-relevant exposure dose studies\nfor hazard identification only, defined as experimental doses >10×\nthe estimated maximum daily human exposure dose (0.22 μg/kg/day),\ni.e., >2.2 μg/kg/day. This classification method provides\nclear\nboundary standards for risk assessment.\n\nEstrogen Receptor α (ERα) is a nuclear receptor that\nbinds estrogens and is predominantly expressed in tissues such as\nthe uterus, ovary (theca cells), testes (Leydig cells), breast, brain,\nand liver. After dimerization, ERα translocates to the nucleus\nwhere it binds to estrogen response elements (EREs) on DNA and recruits\ncoactivators or corepressors,  which subsequently\nregulate the expression of estrogen-responsive genes that play critical\nroles in reproductive processes such as follicular development, ovulation,\nand endometrial proliferation.  Estrogenic\neffects can also occur through ligand-independent activation of ERα,\nwherein cellular signaling pathways induce ERα phosphorylation\nvia protein kinase regulation and second messenger system modifications. \n ,\nMultiple studies have shown that GLY and GBHs, such as Roundup,\ncan activate ERα through direct or indirect mechanisms and disrupt\nestrogen signaling pathways. Dose-dependent phosphorylation at the\nSer118 site, nuclear translocation of ERα, and upregulation\nof estrogen-responsive genes have been observed, which promote the\nproliferation of breast cancer cells (e.g., MCF-7 and T47D cells).  GLY has also been shown to upregulate ERα\nexpression in T47D cells.  ERE-luciferase\nreporter gene assays confirmed its xenoestrogenic activity via ERα-mediated\nmechanisms, as both enhanced ERE transcriptional activity and T47D\ncell proliferation were abolished by treatments with an ERα\nantagonist. \n , \n  Although molecular dynamic simulations\nsuggest that GLY may interact with the ligand-binding domain of ERα\nby forming a complex with zinc ions,  another\nstudy revealed significantly weak binding energy between GLY and ERα\n(−4.10 kcal/mol) compared to 17β estradiol (E2) (−25.79\nkcal/mol), indicating an unstable interaction.  Additionally, the same study reported that IBMX, a cAMP-PKA\nsignaling activator, induced ERE-mediated reporter gene expression,  suggesting that GLY may modulate ERα activity\nvia ligand-independent pathways, such as cAMP-dependent PKA pathway.\nNevertheless, the absence of direct binding evidence and  in\nvivo  validation limit conclusions regarding ligand-independent\nactivation. These studies suggest that ERα is a potential initial\ntarget molecule for GLY/GBH, which may lead to adverse female reproductive\noutcomes such as abnormal endometrial proliferation, uterine lesions,\nand impaired embryonic development.\nAromatase (Cyp19a1, estrogen synthase), a member of the cytochrome\nP450 superfamily, is a key enzyme in estrogen biosynthesis. In the\nspecialized cells of the ovary, hypothalamus, and placenta, aromatase\nplays a crucial role in mammalian reproduction by catalyzing the conversion\nof androstenedione and testosterone to estrone (E1) and E2, respectively.\nIn particular, ovarian aromatase generates both systemic and locally\nactive estrogen. \n , \n  Brain aromatase regulates the\nhypothalamic-pituitary–gonadal (HPG) axis through modulation\nof local estrogen synthesis, subsequently influencing gonadotropin-releasing\nhormone (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\ncan effectively eliminate estrogen biosynthesis.  Much attention has been paid to understanding the regulation\nof the aromatase gene and its role in the development and progression\nof estrogen-dependent diseases such as breast cancer, endometrial\ncancer, and endometriosis.  As a result,\naromatase has been identified as a key molecular target for many environmental\nendocrine disruptors.\nNumerous  in vitro  studies have demonstrated that GLY and GBHs can\ndirectly suppress aromatase activity. Richard et al. first reported\nthat GLY and GBHs dose-dependently inhibited aromatase activity in\nhuman JEG-3 placental cells.  The IC 50  was 0.04% Roundup (equivalent to 0.84 mM GLY), which represents\na concentration lower than that used in typical agricultural applications\n(1–2% Roundup, containing 21–42 mM GLY).  Mechanistically, GLY and GBHs inhibited aromatase\nactivity by binding directly to the active site of the enzyme, as\nevidenced by characteristic spectral changes (type II spectrum) resulting\nfrom interactions between GLY/GBHs and the heme iron atom of aromatase\nin purified aromatase systems.  The data\nalso indicated that GBHs exerted a stronger inhibitory effect on aromatase\nthan GLY alone. The authors hypothesized that adjuvants in GBHs, such\nas polyoxyethylene amine (POEA), significantly improved cell membrane\npermeability and bioaccumulation, thereby increasing intracellular\nbioavailability and enhancing aromatase inhibition through targeted\ndelivery to endocrine active tissues.  Additionally, GLY and GBHs may reduce aromatase activity by downregulating\nthe expression of the  Cyp19  gene.  Benachour et al. further demonstrated that GLY and GBHs\nnot only interact directly with the activity site of aromatase, but\nalso affect its auxiliary enzymesNADPH, a cytochrome P450\nreductase.  This dual mechanism enhances\nthe inhibitory effect of GLY on aromatase in the cellular environment.\nGLY and GBHs exhibited more potent aromatase inhibition in human embryonic\nkidney 293 cells transfected with the aromatase gene than in microsomes,\nsuggesting that the cellular environment may amplify their inhibitory\neffects.  These findings suggest that\nGLY and its commercial formation may disrupt aromatase activity at\nconcentrations below typical agricultural application levels, thereby\ninterfering with estrogen synthesis. Such hormonal disturbances could\npotentially lead to adverse effects on fetal development and reproductive\nhealth.\nAlthough current  in vivo  studies in\nfemale mammals\nmeeting the screening criteria remain insufficient to validate the\naforementioned  in vitro  findings, a recent investigation\non the GLY-induced reproductive toxicity in adult female climbing\nperch has produced results consistent with  in vitro  experimental conclusions. The study demonstrated that GLY binds\nto brain-type aromatase at residues MET424, THR423, and PRO479 (binding\nenergy: −10.685 kcal/mol) and to ovarian-type aromatase at\nresidues ASN479, THR477, among others (binding energy: −10.685\nkcal/mol). All GLY-treated groups (2.6, 3.9, and 7.8 mg/L) exhibited\novarian follicular wall rupture and oocyte atresia, with additional\nvacuolization observed in the low-concentration group (2.6 mg/L).\nNotably,  Cyp19A1A  (ovarian-type) and  Cyp19A1B  (brain-type) expression was significantly downregulated in the highest\nconcentration group (7.8 mg/L GLY).  The\nauthors hypothesize that GLY may impair reproductive function through\naromatase inhibition and disruption of the HPG axis.  Importantly, direct aromatase inhibition by GLY may contribute\nto ovarian dysfunction, hormone dysregulation, and impaired fetal\ndevelopment.\nThe electron transport chain (ETC),\nalso known as the respiratory chain, consists of large protein complexes\n(CI, CII, CIII, CIV, CV) and two mobile electron carriers, ubiquinone\nand cytochrome c (Cytc), located in the inner mitochondrial membrane\ncristae.  Inhibition of the ETC triggers\na cascade of mitochondrial events, including excessive production\nof reactive oxygen species (ROS), impaired oxidative phosphorylation\nleading to reduced ATP synthesis, a decreased ATP/ADP ratio, release\nof Cytc from the mitochondrial cristae, and loss of mitochondrial\nmembrane potential (MMP). \n ,\nPeixoto et al.\nwere the first to demonstrate that 15 mM GBHs directly inhibit mitochondrial\nrespiratory chain complexes II (succinate dehydrogenase, SDH) and\nIII (succinate-Cytc reductase), thereby disrupting ETC function in\nisolated rat liver mitochondria.  This\ninhibition triggered a series of adverse mitochondrial effects, including\nreduced mitochondrial membrane potential, uncoupled oxidative phosphorylation,\nsuppressed ATP synthase activity, and consequent ATP synthesis reduction.\nSubsequent observations revealed mitochondrial swelling (15 mM GBH\ntreatment) and increased membrane permeability (10 mM GBH treatment).\nThe inhibition of ETC complexes has been\nidentified as a key molecular\nmechanism contributing to GLY-induced female reproductive toxicity,\nas evidenced in human-derived  in vitro  models representing\nvarious components of the female reproductive system. Exposure to\nGLY or GBHs significantly suppressed SDH activity in human placental\ncells (JEG-3), human umbilical vein endothelial cells (HUVEC), and\nhuman embryonic kidney cells (HEK 293).  Notably, GLY alone inhibited mitochondrial SDH activity across all\ntested cell types, showing partial inhibition observed at 7.2 g/L\nand significant inhibition at 360 g/L. However, GBH demonstrated more\npotent SDH inhibition at substantially lower concentration (0.5% Roundup,\ncontaining 1.8 g/L GLY equivalent), as formulation adjuvants (e.g.,\nPOEA) enhanced cellular uptake and destabilized mitochondrial membranes,\nthereby potentiating SDH inhibition.  This\nmitochondrial respiratory chain impairment was consistently associated\nwith increased apoptotic cell death in all tested cell lines.\nIn vivo  studies have\nreported that GBH exposure\n(3–10% GLY as Touchdown Hitech, containing 30–100 g/L\nGLY equivalent) inhibits mitochondrial SDH activity in  Caenorhabditis\nelegans  and reduces respiratory efficiency.  However, research on GLY-induced placental mitochondrial\ndysfunction in mammals remains limited. Bai et al. reported that high-dose\nGBH exposure (100 mg/kg) during pregnancy significantly upregulated\nthe mRNA expression of mitochondrial fission gene  Fis1 , fusion gene  MFN2  and  SDHA  in\nboth porcine placenta and piglet jejunums, consequently impairing\nplacental angiogenesis and mitochondrial function. \n , \n  These findings suggest an evolutionarily conserved mechanism of\nmitochondrial dysfunction across cell types and underscore the potential\nrole of ETC impairment in GLY-induced female reproductive toxicity.\n\nReactive oxygen species (ROS), including\nsuperoxide anion radical (O 2–• ), hydrogen\nperoxide (H 2 O 2 ), hydroxyl radical (OH • ), and singlet oxygen, are crucial regulators of cell energy metabolism\nand proliferation. While physiologically essential, ROS imbalance,\nparticularly excessive accumulation, can induce cellular dysfunction\nand irreversible damage.  Both exogenous\nstressors and normal endogenous cellular processes, notably mitochondrial\nETC activity, contribute to ROS generation.  Evidence indicates that GLY induced ROS overproduction through ETC\nimpairment  and mitochondrial dysfunction,\nresulting in DNA damage and cell cycle arrest in MCF-7 (a hormone-dependent\nhuman breast cancer cell line) and MDA-MB-468 (a hormone-independent\nhuman breast cancer cell line).  Studies\ndemonstrate that GLY chelates intracellular zinc in mice oocytes,\ncompromising antioxidant defenses and exacerbating mitochondrial dysfunction,\nwhich dose-dependently elevates ROS levels. \n , \n  These alterations impair oocyte competence and postfertilization\nembryo development. In addition, GLY exposure increases uterine ROS\nlevels in piglets, alters uterine and ovarian tissue morphology and\nultrastructure, and disrupts hormone balance.  Notably, even at concentrations below agricultural application levels\n(0.9 ppm Roundup, containing 5.33 μM GLY), GBH induced apoptosis\nvia ROS-mediated pathways, compromising early bovine embryonic development.\nOxidative stress, characterized by an\nimbalance between ROS production and antioxidant defense capacity,\ninvolves key enzymes including superoxide dismutase (SOD), catalase\n(CAT), glutathione peroxidase (GPx), and glutathione (GSH),  which cooperatively maintain the reduction–oxidation\nhomeostasis of cells. These enzymatic systems, crucial for cellular\nprotection against oxidative damage, can serve as biomarkers of oxidative\nstress induced by exogenous substances. GLY exposure increased ROS\nlevels, eliciting compensatory SOD/CAT activation in mouse ovaries, \n , \n  piglets uterus,  and oocytes of mice\nand cattle. \n , , \n  Prolonged exogenous oxidative effects can induce mitochondrial ROS\naccumulation beyond cellular clearance capacity, leading to cumulative\noxidative damage that impairs mitochondrial function, reduces antioxidant\nsystem efficiency, and exacerbates cellular oxidative stress.  Treated with high-dose GLY (105 μg/kg\nbw GLY) for 28 days impaired antioxidant function of rat’s\novaries, by suppressing ovarian CAT and SOD activity and downregulated\nglutathione reductase (Gsr) gene expression in GLY-exposed female\nrats, concomitant with endocrine disruption and impaired folliculogenesis.  Similar dose-dependent decreases in CAT, SOD,\nGPx, and GSH levels, accompanied by apoptosis and steroidogenesis\ndysregulation, were observed in rat  and\nbovine  ovaries following GLY exposure.\nGBH-induced antioxidant depletion exacerbates oxidative and endoplasmic\nreticulum stress, mitochondrial dysfunction, and apoptosis in placental\ncells, leading to developmental abnormalities in fetal mice  and piglets. \n In vitro  studies corroborated these findings, demonstrating\nthat GLY exposure during porcine oocytes meiosis elevated intracellular\nROS levels while disrupting mitochondrial dynamics and calcium homeostasis,\nthe expression of antioxidant-related genes ( SOD1 ,  GPx ,  CAT ) was observably suppressed,\nsuggesting oxidative stress in mitochondria and cytoplasm, ultimately\ncompromising oocyte quality.  Taken together,\nGLY-induced female reproductive toxicity primarily involves ROS overproduction,\noxidative stress, and apoptotic pathway activation, manifesting as\novarian dysfunction, embryonic developmental abnormalities, and hormonal\ndysregulation, which are the most frequently observed key molecular\nevents in GLY-induced toxicity.\nDNA damage,\nincluding nucleotide modifications, single-strand breaks (SSBs), and\ndouble-strand breaks (DSBs), occurs during cellular processes and\ncan be directly induced by endogenous or exogenous stressors (e.g.,\nROS, chemical agents, and ionizing radiation). \n , \n  GLY-mediated female reproductive toxicity may involve two DNA damage\nmechanisms, including direct interference with DNA-associated proteins\nand repair machinery and/or ROS-mediated indirect genotoxicity. Experimental\nevidence from ER-dependent genotoxicity studies revealed that HEC1A\ncells (ER-positive endometrial cancer cell line) showed increased\nsensitivity, characterized by decreased cell viability, elevated DNA\nfragmentation, mitochondrial depolarization, and early apoptosis.  However, MDA-MB-231 cells (ER-insensitive breast\ncancer cell line) exhibited less toxicity but still detectable DNA\ndamage. \n , \n  Mechanistic studies indicated that GBHs\nmay dysregulate DNA damage repair pathways, particularly the base\nexcision repair (BER) system, through downregulation of key genes\n(e.g.,  OGG1 ,  XRCC1 ).  This suppression may occur through altered oxygen\nconsumption, ROS elevation, and subsequent hypoxia which may promote\nDNA damage, G1/S phase arrest, and subsequent apoptosis.  In mouse oocytes, GLY-induced ROS overproduction\nwas associated with oxidative DNA damage (particularly DSBs), impairing\nmaturation rates and developmental potential while triggering autophagy\nand premature apoptosis.\nLipid\nperoxidation is a process that involves the oxidative degradation\nof membrane lipids, compromising the structural integrity of the cellular\nand organelle membranes. This pathological process is primarily driven\nby ROS, which target the unsaturated carbon–carbon bonds within\nfatty acids that constitute lipids. Lipid peroxidation may disrupt\ncellular homeostasis ultimately leading to membrane destabilization\nand dysfunction.  Accumulating evidence\nsuggests that GLY and GBHs induced female reproductive toxicity via\noxidative stress-induced lipid peroxidation. Studies described in\nsection 4.4.1 demonstrated that GLY-induced oxidative stress, manifested\nby elevated ROS levels, was invariably accompanied by lipid peroxidation\nin ovarian and uterine tissues. This was evidenced by increased malondialdehyde\n(MDA) levels. \n , , \n  Lipid peroxidation may impair membrane protein function and increase\nmembrane permeability, leading to mitochondrial swelling and the collapse\nof membrane potential. These effects may compromise organelle membrane\nintegrity, potentially causing nuclear and mitochondrial membrane\ndestabilization in porcine uterine cells, structural disorganization\nof mitochondrial endoplasmic reticulum in porcine oocytes, and endoplasmic\nreticulum stress in mouse placenta following exposure to GLY and/or\nGBH. \n , \n  The consequent structural damage may activate\napoptotic pathways via Bax/Bcl-2 imbalance and caspase-3/9 activation,\nultimately causing ovarian follicular atresia and placental hypoplasia. \n , \n  These findings indicate that GLY exposure may cause female reproductive\ntoxicity through oxidative stress-mediated lipid peroxidation, which\ndisrupts cellular homeostasis and triggers apoptotic cascades. This\npathological process is characterized by upstream redox imbalance,\ndownstream organelle dysfunction, and terminal ovarian failure and\ndevelopmental abnormalities.\nThe endometrium serves as a primary target tissue for estrogen,\norchestrating critical physiological functions in embryo implantation\nand maintenance through tightly regulated hormonal signaling. Estrogen\nexerts its reproductive effects predominantly via two classical nuclear\nreceptors, ERα and estrogen receptor β (ERβ), which\ncoordinate cyclical endometrial proliferation and differentiation.  Aberrant ER expression patterns in the endometrium\nare associated with various pathological conditions including endometriosis,\nendometrial hyperplasia, and endometrial cancer.  Mechanistic studies have demonstrated that dysregulation\nof ER subtype stoichiometry (ERα/ERβ ratio) and spatiotemporal\nexpression dynamics may contribute to several pathogenic processes,\nincluding uncontrolled cellular proliferation, impaired apoptotic\nregulation, and compromised decidualization, all of which represent\ncharacteristic features of endometrial pathophysiology.\nIn vitro  studies using\na human endometrial adenocarcinoma cell line (Ishikawa) demonstrated\nthat GBH enhanced cell migration and invasion, concomitant with the\nsuppression of epithelial-mesenchymal transition (EMT) markers such\nas E-cadherin mRNA. These pro-metastatic effects were fully blocked\nby the ER antagonist fulvestrant, suggesting that GBH may promote\nmalignant transformation and metastasis of endometrial cancer via\nER-dependent mechanisms. \n In vivo  developmental exposure to GBH disrupted uterine homeostasis, inducing\nendometrial hyperplasia in juvenile rats and predisposing adult rats\nto subsequent uterine pathologies, such as adenomyosis. These effects\ncorrelated with aberrant  Hoxa10  epigenetic silencing\nand sustained estrogen dominance via elevated E2/P4 ratios.  Parallel studies showed that perinatal GBH exposure\ndysregulated uterine ER isoform dynamics, downregulating epithelial\nERα while upregulating stromal ERβ and progesterone receptor.  Receptor imbalance is associated with endometriosis\nand endometrial carcinogenesis.  In addition,\nstudies suggest that GLY and/or GBHs upregulated ERα gene expression\nepigenetically through hypomethylation and histone modification shifts\nof the ERα promoter.  ERα\noverexpression may contribute to embryo implantation failures.  These findings implicate GLY/GBH as ER-modulating\nendocrine disruptors, driving female reproductive toxicity through\nEMT activation, epigenetic dysregulation of developmental genes, and\nhormonal imbalance, ultimately promoting uterine dysfunction and carcinogenic\nprogression.\nMitochondrial dysfunction is a central mechanism in GLY-induced female\nreproductive toxicity, primarily driven by oxidative stress and impaired\nETC activity. As described in  Sections  \n  and  4.1.1 ,\nGLY directly inhibits ETC complexes II and III, leading to excessive\nROS production and oxidative stress. Mitochondria are both the primary\nsource of ROS within cells and the most vulnerable organelle to ROS-induced\ndamage.  Excessive ROS production disrupts\nmitochondrial function through multiple pathways, including oxidizing\niron–sulfur clusters and heme groups within ETC complexes I,\nIII, and IV, leading to impaired electron transport and reduction\nof ATP synthesis. Increased electron leakage may further contribute\nto a regenerative cycle known as ″ROS-induced ROS release″.\nROS also directly affect mitochondrial DNA (mtDNA), causing base\ndeletion, strand breaks, and mutations. These changes may affect the\nexpression of coding genes of the mitochondrial respiratory chain\ncomplex, further disrupting oxidative phosphorylation. The inner mitochondria\nmembrane, rich in unsaturated fatty acids, is particularly susceptible\nto ROS-induced lipid peroxidation, which compromises the membrane\nintegrity. In addition, lipid peroxidation products can activate the\nmitochondrial permeability transition pore (mPTP), triggering the\nrelease of Cytochrome c and other pro-apoptotic factors, which initiate\napoptosis. \n ,\nSubstantial evidence supports\nthe role of mitochondrial dysfunction\nin GLY-induced female reproductive toxicity. For instance, studies\non GLY-exposed porcine oocytes observed elevated ROS, reduced mitochondrial\nDNA copy numbers, and extensive mitochondrial damage that is evidenced\nby reduced distribution of mitochondria in the oocyte cortex, decreased\ncopy number of mtDNA, and downregulated expression of  PGC1α  and  ATP5B  genes.  These\nchanges were associated with impaired meiotic progression, decreased\noocyte maturation rates, and reduced oocyte quality. Similarly, in\nbovine and mice oocytes, GLY exposure led to abnormal intracellular\nROS accumulation, decreased mitochondrial membrane potential, and\nupregulation of apoptosis-related genes ( Caspase-3/Caspase-4,\nBAX ), initiating early apoptosis and autophagy. \n , , \n \n In vivo  murine\nstudies have shown that GLY-induced oxidative stress and mitochondrial\ndysfunction are associated with multiple pathological effects, including\novarian cell apoptosis, reduced ATP production, endometrial glandular\natrophy, follicular atresia, as well as dyshomeostasis of thyroid\nhormone and hypothalamic-pituitary-ovarian (HPO) axis hormones. \n , \n  Furthermore, GBH-induced mitochondrial dysfunction in porcine placental\ntissues was linked to impaired vascular formation, barrier integrity,\nand nutrient transport, potentially affecting neonatal development.  In summary, mitochondrial dysfunction may play\na critical role in GLY-induced female reproductive toxicity, linking\nupstream oxidative stress and ETC impairment to downstream pathological\noutcomes, such as apoptosis, oocyte maturation defects, and developmental\nabnormalities.\nEpigenetic modifications, defined as heritable changes in gene\nexpression without alterations to the underlying DNA sequence,  include DNA methylation, histone modification,\nnucleosome assembly/remodeling, and noncoding RNA-mediated regulation.  Among these, DNA methylation and histone modifications\nmay be central mechanisms in GLY-induced female reproductive toxicity.\nDNA methylation involves the covalent addition of methyl groups to\ncytosine residues within CpG dinucleotides and is catalyzed by DNA\nmethyltransferases (DNMTs). Modifications predominantly occur in gene\npromoter regions, where hypermethylation typically suppresses transcription. \n ,\nEmerging evidence indicates that GLY and GBHs may disrupt\nfemale reproductive function through epigenetic reprogramming, particularly\nvia DNA methylation and histone modification. Alterations of this\nnature can impair key gene networks essential for endometrial receptivity\nand uterine development, ultimately leading to adverse outcomes, such\nas embryo implantation failure and estrogen-dependent disorders. Lorenz\net al.  showed that gestational and lactational\nexposure to GLY at a dose of 2 mg/kg bw/day significantly upregulated\nDNA methyltransferase DNMT3a in rats. This upregulation was associated\nwith hypermethylation at CpG islands within the promoter and regulatory\nregions of  Hoxa10 , a critical regulator of endometrial\nreceptivity.  Hypermethylation correlated\nwith significant downregulation of  Hoxa10  mRNA, and\nimpaired embryo implantation. Additionally, 2 mg/kg/day GLY exposure\nin rats induced the same aberrant epigenetic modifications in the  Hoxa10  gene, resulting in hyperplasia of the endometrium\nand myometrium.  Similarly, Almiron et\nal. observed hypermethylation at a CpG island in the  Lif  promoter in rats following exposure to either 3.8 mg GLY/kg/day\nGBH or 3.9 mg GLY/kg/day pure GLY, which resulted in a 60% reduction\nin  Lif  mRNA levels. \n Lif  encodes a cytokine essential for embryo-uterine crosstalk,\nand its suppression compromises endometrial receptivity. Furthermore,\na recent study showed that prenatal GBH exposure downregulated the\nmRNA levels of  Dnmt1  and  Dnmt3b  genes\nin the jejunum of offspring piglets, which was associated with diminished\nDNA methylation, which may impair intestinal development and barrier\nfunction in newborn piglets.\nHistones,\nparticularly the H3 variant, play a pivotal role in epigenetic\nregulation by organizing DNA into nucleosomes and modulating chromatin\naccessibility.  PTMs of histone tails,\nsuch as methylation, acetylation, and phosphorylation, regulate chromatin\ncompaction states and transcriptional activity.  For example, methylation of histone H3 at lysine 4 (H3K4me1/me2/me3),\nlysine 36 (H3K36me), or lysine 79 (H3K79me) is associated with open\nchromatin and transcriptional activation.  In contrast, di- or trimethylation of H3K9 (H3K9me2/me3) and H3K27\n(H3K27me2/me3) promotes heterochromatin formation and gene silencing.  The functional consequences of histone methylation\ndepend on both the specific lysine residue modified and the degree\nof methylation (mono- vs polymethylation). These modifications are\ndynamically regulated by histone methyltransferases (HMTs) and demethylases\n(HDMs), maintaining a balance critical for epigenetic homeostasis.\nGLY/GBHs elevated repressive histone modifications, such as H3K27me3\n(catalyzed by EZH2) and H3K9me3, in the  Hoxa10  and  Lif  regulatory regions. These modifications alter chromatin\naccessibility, further silencing genes critical for implantation. \n , \n  Lorenz et al. demonstrated that GBH reduced DNA methylation at the\nERα-O promoter and increased activating histone marks (e.g.,\nH4Ac↑, H3K27me3↓), leading to a 2.5-fold increase in  ERα  expression.  ERα\nhyperactivation can cause uterine hyperplasia and estrogen-dependent\npathologies, including adenomyosis and atypical endometrial hyperplasia.\nIntegrating these findings, MIEs may involve\nbinding of GLY/GBH\nto ERα or antioxidant enzymes, activating DNMTs/EZH2, and driving\nDNA and histone hypermethylation. Alternatively, exposure may cause\noxidative stress, disrupting the balance between histone acetyltransferase\n(HAT) and histone deacetylase (HDAC). These KEs may perturb gene networks\n( Hoxa10 ↓ , Lif ↓ , ER α↑), culminating in AOs at both tissue\n(e.g., impaired implantation) and organismal (e.g., infertility, neoplasia)\nlevels.\nThe spindle\napparatus, a dynamic cytoskeletal structure essential for eukaryotic\ncell division, plays a vital role in mitosis and meiosis by ensuring\nprecise chromosome separation.  In mammalian\noocytes, which lack centrioles and centrosomes,  meiotic spindle formation assembly depends on microtubule-organizing\ncenters (MTOCs) that substitute for conventional centrosomes.  Proper spindle assembly and organization are\ncritical to normal chromosome dynamics, including the accurate alignment\nof chromosomes during metaphase and their segregation into daughter\ncells during anaphase.  Maintaining normal\nspindle structure and function is indispensable for ensuring the fidelity\nof chromosome segregation during meiosis, particularly during the\nphases of spindle assembly and chromosome alignment.\nEvidence\nsuggests that exposure to GLY and GBHs induces female reproductive\ntoxicity by causing spindle abnormalities and chromosomal dysregulation\nduring oocyte meiosis. GLY/GBHs trigger intracellular oxidative stress\nand redox imbalance, potentially initiating a cascade of cellular\nperturbations, including the suppression of p-MAPK expression, which\nplays a critical role in regulating microtubule dynamics and spindle\nassembly. \n , , \n  Mouse oocytes\nexposed to 50–300 μM GLY exhibited profound cytoskeletal\ndisorganization characterized by shortened or disrupted spindle fibers,\nabnormal MTOC formation, and chaotic chromosomal alignment. The concentration-dependent\ndegradation of spindle structure suggests cumulative damage to microtubule\npolymerization processes, which directly impairs meiotic progression.  This is evidenced by reduced polar body extrusion\nrates and metaphase II arrest, indicating compromised chromosomal\nsegregation fidelity.  In addition, DNA\ndouble-strand breaks, caused by other MIEs such as oxidative stress,\nlikely exacerbate chromosomal instability during forced segregation\nattempts.\nMitochondrial dysfunction,\nanother consequence of oxidative stress,\nfurther diminishes oocyte quality by depleting ATP reserves essential\nfor spindle checkpoint surveillance and apoptosis regulation.  The downstream developmental consequences are\nsevere and diverse. Impaired cytoplasmic maturation in GLY/GBH-exposed\noocytes resulted in reduced fertilization competence and compromised\nembryonic potential, as demonstrated by reduced 2-cell embryo formation. \n , \n \n In vivo  studies confirmed these findings, with\nGLY/GBH-exposed mice showing parallel declines in oocyte maturation\nrates and early embryonic developmental capacity.\nCollectively, these findings outline a pathogenic\npathway in which\noxidative stress-induced MAPK suppression disrupts spindle morphogenesis,\nleading to chromosomal missegregation, meiotic failure, and ultimately\nreduced reproductive success.\nIn the\nstudies of GLY/GBHs-induced female reproductive toxicity, cell death\nrepresents an end point of cytotoxic effects at the cellular level.\nEvidence from both  in vivo  and  in vitro  studies indicates that GLY or GBH exposure reduced the cellular\nviability in several female reproductive cell types, including oocytes,\ngranulosa cells, placental cells, embryonic cells, and endometrial\ncarcinoma cells. Notably, the specific modes of cell death vary significantly\ndepending on exposure conditions and cell type. Cell death mechanisms\nare broadly classified into programmed cell death (PCD) and nonprogrammed\ncell death (NPCD). Unlike uncontrolled necrotic processes, which typically\ninduce inflammatory responses, PCD involves genetically regulated,\nself-executing pathways for active cell termination. The predominant\nPCD modalities associated with GLY toxicity include apoptosis and\nautophagy. Activation of apoptosis through the intrinsic mitochondrial\npathway is the most commonly reported cytotoxic mechanism of GLY exposure.\nGLY and GBHs dose-dependently elevated intracellular ROS levels and\nsubsequently upregulate pro-apoptotic markers (Bax, caspase-3/9) while\nsuppressing antiapoptotic Bcl-2 expression. \n , , \n  The critical shift in the Bax/Bcl-2 ratio\ntoward the initiation of apoptosis resulted in collapse of the mitochondrial\nmembrane potential, release of Cytc, and activation of the apoptotic\ncascade. Characteristic apoptotic markers, including annexin-V and\nelevated caspase-3/9 expression, have been observed in GLY-exposed\noocytes and embryonic cells. \n ,\nIn addition to\napoptotic activation, GLY exposure induced autophagic responses, potentially\nas a cytoprotective adaptation to cellular stress. This compensatory\nmechanism is supported by upregulation of autophagy-related genes\n( LC3, Beclin-1, ATG12 ) and corresponding protein\nmarkers in reproductive tissues, suggesting enhanced removal of damaged\ncellular components. \n , , \n  However, prolonged oxidative stress can turn this adaptive mechanism\ninto a maladaptive response, exacerbating mitochondrial dysfunction\nand contributing to reproductive impairment through excessive autophagic\nactivity.\nHigh-dose or prolonged GLY exposure promotes necrotic\ncell death\nprogression. Subchronic exposure to GLY at a high dose of 315 mg/kg\nhas been shown to induce characteristic necrotic alterations in rats,\nincluding oocyte nuclear pyknosis. \n In vitro , the cytotoxic effects of GBHs on three human cell\ntypes (HUVECs, HEK293, and JEG-3) revealed that GBH exposure caused\ncell membrane damage within 24 h, as evidenced by the release of cytosolic\nadenylate kinase (AK), a marker of cell membrane rupture associated\nwith late-stage necrosis.\nOverall,\ncell injury and death induced by GLY/GBHs through apoptosis,\nautophagy, and necrosis may represent key mechanisms underlying GLY-induced\nfemale reproductive toxicity. These molecular injuries can lead to\nfunctional impairments in reproductive tissues, with ovaries exhibiting\nfollicular atresia, granulosa cell necrosis, and oocyte vacuolization,\nwhile placental dysfunction may result in growth-restricted fetuses. \n ,\nThe disruption of hormonal homeostasis may also contribute to GLY-induced\nfemale reproductive toxicity, serving as both a consequence and an\namplifier of cellular dysfunction across multiple regulatory levels.\nGLY and GBHs can impair steroidogenesis through oxidative stress-mediated\npathways and/or the inhibition of aromatase, directly targeting ovarian\ngranulosa cellsthe primary site of 17β-estradiol (E2)\nand progesterone (P4) synthesis.  Studies\nacross species, including porcine, bovine, and murine models, have\nconsistently demonstrated GLY’s capacity to suppress granulosa\ncell proliferation and metabolic activity while disrupting steroidogenic\nenzyme function. For example, in porcine granulosa cells, 0.2–16\nμg/mL GLY significantly reduced E2 secretion while paradoxically\nincreasing P4 levels, indicating dysregulation of steroidogenic enzyme\ncascades.  Similarly, bovine granulosa\ncells exposed to GLY at 0.5–5 μg/mL exhibited dose-dependent\ninhibition of E2 synthesis without affecting P4 secretion, likely\ndue to direct interference with aromatase activity.  In addition, another  in vitro  study showed\nthat GBH (containing 10 μg/mL GLY) exposure significantly inhibited\ngranulosa cell proliferation and steroid production (E2 and P4).  Selective disruption of E2 production was associated\nwith GLY-induced oxidative stress and apoptosis, which may drive steroidogenic\ndysfunction in murine ovaries, ultimately leading to hormonal imbalance\nand reduced fertility.  However, higher\nconcentrations of GBHs (10–300 mg/mL) caused broader suppression\nof both E2 and P4 in bovine models, suggesting concentration-dependent\ntoxicity that may overwhelm compensatory mechanisms.  Reduced E2 levels disrupted the positive feedback loop\nnecessary for follicular selection and ovulation, while abnormal P4\ndynamics, as observed in porcine oocytes, correlated with diminished\nblastocyst formation rates and compromised embryonic viability.  These findings collectively position disrupted\nsteroid hormone synthesis as an upstream key event that triggers downstream\nconsequences, such as follicular atresia (via loss of E2-mediated\nsurvival signals) and ovulation failure (due to aberrant P4-regulated\nluteinization). \n ,\nParadoxically, some  in vivo  studies by Lorenz et al. and Guerrero et al. reported\nelevated serum E2 levels and decreased P4, suggesting complex systemic\nfeedback mechanisms that may override local ovarian dysfunction. \n , \n  This apparent contradiction likely arises from multilevel endocrine\ndisruption spanning the HPO axis. GLY exposure induced upstream dysregulation\nof GnRH and pituitary gonadotropins, as demonstrated by the downregulation\nof GnRH gene expression and upregulation of FHSβ and LHβ\nexpression.  Compensatory increases in\nLH secretion could transiently stimulate residual ovarian steroidogenesis\nwhile simultaneously accelerating follicular depletion through excessive\nluteinization. Furthermore, the estrogenic activity of GLY via ERα\nupregulation may establish pseudohyperestrogenic conditions despite\nimpaired ovarian E2 production.  The observed\nserum hormone fluctuations likely reflect disrupted negative feedback\nmechanisms, wherein ovarian resistance to gonadotropins, combined\nwith direct hypothalamic-pituitary effects and brain aromatase inhibition,\ngenerates oscillatory hormonal patterns.\nThe divergent P4 responses\nobserved between granulosa cells and\nwhole-organism studies may be attributed to differences in the temporal\nand spatial dynamics. Acute  in vitro  exposure primarily\naffected the steroidogenic capacity of luteinized granulosa cells,\nwhereas chronic  in vivo  exposure additionally impacts\ncorpus luteum formation and maintenance, as well as extra-ovarian\nsteroid metabolism.  GLY-induced disruption\nof thyroid axis could indirectly modulate sex hormone binding globulin\n(SHBG) levels, thereby altering hormone bioavailability.  Oxidative stress emerges as a unifying upstream\nmechanism, which may compromise mitochondrial function in steroidogenic\ncells while activating stress-responsive signaling pathways that dysregulate\nHPOA communication. Downstream consequences include impaired folliculogenesis,\nreduced oocyte competence,  and uterine\nreceptivity defects mediated by altered implantation-related genes\n( Hoxa10, Lif ). These multifaceted disruptions may\ncollectively contribute to diminished reproductive capacity across\nmultiple biological levels.\nOocyte maturation is a critical process in the\nfemale reproductive system involving the progression of oocytes from\nthe germinal vesicle stage to metaphase II, culminating in the release\nof a mature ovum capable of fertilization. This process is essential\nfor successful reproduction as it ensures the genetic integrity and\ndevelopmental potential of the oocyte. GLY and GBHs have been shown\nto impair oocyte maturation and ovulation across multiple mammalian\nmodels through interconnected molecular mechanisms and endocrine disruptions.\nChronic low-dose GLY exposure (2 mg/kg/day for 10 weeks beginning\nat 7 weeks of age) may selectively deplete secondary follicle populations\nvia oxidative stress-mediated mechanisms, demonstrating stage-specific\nvulnerability during follicular maturation.  Exposure to GLY/GBHs induces oxidative stress as a primary upstream\nevent, which may trigger mitochondrial dysfunction, DNA damage, and\nthe subsequent activation of apoptosis and autophagy pathways in oocytes.\nThis cascade of cellular stress responses may induce meiotic arrest,\nas evidenced by decreased germinal vesicle breakdown (GVBD) rates\nand diminished polar body extrusion (PBE) efficiency across multiple\nspecies, including mice ( in vivo  treatment with 250\nmg/kg/day GLY), cattle ( in vitro  treatment with 50\nmM GLY), and pigs ( in vitro  treatment with 400 μmol/L\nGLY). \n , , \n  Compromised\nmeiotic progression may further be exacerbated by structural disorganization\nof MTOCs and chromosomal misalignment, coupled with zinc depletion,\na critical regulator of oocyte maturation.  Additionally, GLY/GBHs reduce E2 and P4 production in granulosa\ncells, which may contribute to meiotic arrest and disrupt the final\nstages of follicular maturation and ovulation. \n , \n  Ren et al.  observed increased atretic\nfollicles and decreased mature follicles in mice exposed to 5 g/L\nGLY in drinking water from the gestational day (GD) 1 to GD 19, along\nwith ovarian stromal fibrosis. These changes were associated with\na significant decline in serum P4 levels and a paradoxical increase\nin estrogen levels. Hormone imbalance may further lead to follicle\natresia and reduced follicle surface area, as reported in other studies.  Collectively, these toxicological and hormonal\nalterations establish a microenvironment wherein GLY exposure potentially\ntriggers oxidative stress as an upstream initiating event, subsequently\ninducing meiotic arrest via impaired germinal vesicle breakdown and\npolar body extrusion.\nOvarian reserve, defined as the number of oocytes within\nthe ovary, declines over time. The finite pool of oocytes available\nfor maturation and fertilization determines the length of a female’s\nreproductive lifespan.  An intact follicle\npool is essential for female fertility, and disruption of its formation\ncan lead to subfertility or infertility. Emerging cross-species evidence\nidentifies ovarian reserve depletion as a critical aspect of GLY-induced\nfemale reproductive toxicity mediated through molecular perturbations\nof folliculogenesis and endocrine dysregulation. For instance, Alarcon\net al. revealed that 1 mg/kg/bw/day GBH exposure disrupts follicular\ndevelopment by downregulating key regulatory molecules, including\nAMH, BMP15, and FSHR, while impairing estrogen and progesterone receptor\nsignaling pathways in peripubertal ewes.  This molecular interference was associated with a pathological feedback\nloop in which reduced AMH expressiona biomarker of ovarian\nreserve, coincides with primordial follicle pool depletion and compromised\nfollicle recruitment.\nUpstream endocrine disruption may further\namplify follicular depletion. GBHs induce paradoxical steroidogenic\nshifts characterized by elevated levels of ovarian Hsd3b1 expression,\npotentially mediated through oxidative stress. This hormonal imbalance\nmay promote premature luteinization of developing follicles, as evidenced\nby increased corpus luteum formation concurrent with diminished primary\nfollicle counts.  Simultaneously, GLY-induced\noxidative stress may disrupt HPOA homeostasis, leading to endometrial\ngland atrophy and ovarian follicular shrinkage in prepubertal swine.  These structural alterations may synergize with\nmolecular disruptions that impair follicular nourishment and oocyte-granulosa\ncell communication. The downstream reproductive consequences may include\ndiminished pregnancy success rates,  reflecting\nboth quantitative loss of ovarian reserve and qualitative impairment\nof remaining follicles.\nEndometrial hyperplasia (EH)\nis characterized by abnormal proliferation of endometrial glands relative\nto the stroma, resulting in an increased gland-to-stroma ratio compared\nto normal proliferative endometrium,  and is strongly associated with prolonged estrogenic stimulation.  Accumulating evidence suggests that EH serves\nas a key event in GLY/GBHs-induced female reproductive toxicity, linking\nmolecular disturbances to pathological progression. As previously\ndiscussed, GLY may initiate endocrine disruption through direct activation\nof ERα and subsequent estrogen signaling pathways, leading to\na significant hormonal imbalance characterized by an increased E2/P4\nratio.  This establishes a hyperestrogenic\nmicroenvironment that can promote endometrial proliferation. \n , \n  Sustained proliferative stimulus is measured by increased uterine\nwall thickness, luminal epithelial hyperplasia, and glandular abnormalities,\nincluding subgland formationa recognized precursor to estrogen-induced\nprecancerous lesions. \n , , , \n  Collectively, these findings\nsuggest that EH may serve as both a biomarker of GLY-induced endocrine\ndisruption and a functional link between MIEs and the downstream neoplastic\nprogression in endometrial carcinogenesis.\n\nPremature ovarian insufficiency (POI) is characterized by insufficient\novarian sex hormone production and a reduced ovarian reserve, leading\nto a rapid decline in ovarian function and early menopause in women.\nFindings from mammalian studies on GLY-induced reproductive toxicity\nare consistent with POI. Ren et al.  observed\nsignificant changes in ovarian morphology and hormone levels in mice\nexposed to 5 g/L GLY in drinking water, consistent with POI pathology.\nSpecifically, they reported an increase in atretic follicles, a decrease\nin mature follicles, and ovarian stromal fibrosis.  Molecular analyses revealed upregulated LHR expression\nand downregulated FSHR expression in ovarian tissue, potentially resulting\nin a pathological feedback loop that exacerbated ovarian dysfunction.\nFurther supporting this association, Novbatova et al. demonstrated\nthat chronic low-dose GLY exposure (2 mg/kg/day for 10 weeks beginning\nat 7 weeks of age) significantly reduced the population of secondary\nfollicles in mouse.  This selective follicular\ndepletion was associated with a decline in pregnancy success rates\nfrom 75% to 55%, highlighting the detrimental impact of GLY on reproductive\noutcomes.\nUterine (endometrial) adenocarcinoma (UA), the most prevalent form\nof uterine cancer, originates from the epithelial cells lining the\ninner layer of the uterus (endometrium) and is classified as type\nI (estrogen-dependent) or type II (nonestrogen-dependent) UA.  Type I UA, the most common subtype, has been\nimplicated in studies of GLY-induced female reproductive toxicity.\nGuerrero et al. reported that exposure to GBHs (2 mg of GLY/kg/day)\nduring the developmentally sensitive postnatal period [postnatal day\n(PND) 1–7] induced multiple uterine lesions in aged rats (PND\n600).  The observed pathologies comprised\nadenomyosis, formation of estrogen-induced precancerous daughter glands,\natypical endometrial hyperplasia (a UA precursor), and uterine leiomyomas.  As discussed in  Section  \n , GLY may trigger endometrial epithelial\nproliferation through hormonal imbalance and uterine structural changes\nmediated by estrogen signaling pathways, leading to estrogen-dependent\nprecancerous lesions such as adenomyosis and endometrial atypical\nhyperplasia. \n , \n  Similarly, GLY-induced ERα\nupregulation was accompanied by depletion of tumor suppressors ( PTEN  and  p27 ) in hyperplastic endometrium\n- hallmarking endometrial cancer initiation.  Furthermore, Gastiazoro et al. documented the progression from benign\nhyperplasia to atypical endometrial hyperplasia and adenomyosis, highlighting\nthe potential of GBHs to induce EMT in endometrial carcinoma cells,\ncharacterized by E-cadherin suppression and enhanced migratory capacity.  Notably, AOP 503 in the AOP-wiki describes a\ncomplete pathway in which ERα activation promotes epigenetic\nmodification of proliferative factors, ultimately leading to endometrial\ncancer. Given that GLY activates ERα and induces epigenetic\nprocesses associated with similar AOs, further research is needed\nto determine whether GLY contributes to endometrial cancer through\nthe direct epigenetic modification of proliferative factors.\nImpaired ovarian function and reproductive system diseases, such\nas endometrial cancer, significantly reduce the likelihood of successful\npregnancy and severely compromise fertility. \n , , , , \n  As previously discussed, exposure to GLY or GBHs\ncan lead to outcomes such as POI and UA, thereby adversely affecting\nreproductive capacity. Ingaramo et al. found that the number of resorption\nsites was significantly increased on GD 19, after the newborn female\nrats were exposed to 2 mg/kg/day GBH on PND1, 3, 5, and 7.  Lorenz et al.  reported\nthat exposure to GBHs (350 mg GLY/kg bw/day) increased the rate of\nimplantation failure in F1 generation rats during early pregnancy.\nFurthermore, Milesi et al.  observed\nthat perinatal exposure to GBHs resulted in a higher rate of embryo\nimplantation failure in rats. F2 generation offspring in the GBH-exposed\ngroup exhibited reduced body weight and body length, while a high-dose\nGBH (200 mg of GLY/kg of bw/day) exposure group led to structural\ncongenital malformations in F2 fetuses, potentially causing severe\nlong-term health consequences.\n\nThis review\nsystematically elucidated the mechanisms by which GLY\nmay cause female reproductive toxicity across multiple biological\nlevels by using the AOP framework. By integrating core concepts such\nas MIEs, KEs, and AOs, this review highlights toxicological end points\ntriggered by GLY and its formulated herbicides (GBHs) in the female\nreproductive system.\nThe AOP framework, constructed based on\nthe AOP-wiki database and\npublicly available data, identifies three primary MIEs through which\nGLY may cause toxicity: (1) activation of ERα, (2) inhibition\nof aromatase activity, and (3) disruption of mitochondrial ETC complexes.\nThese MIEs may trigger a cascade of KEs at the molecular and cellular\nlevels, including increased ROS levels, oxidative stress, DNA damage,\nlipid peroxidation, disruption of estrogen receptor signaling pathways,\nmitochondrial dysfunction, abnormal epigenetic modifications, spindle\napparatus defects, and altered chromosome dynamics, ultimately leading\nto cell death. At the tissue/organic level, critical events included\nhormonal homeostasis imbalance (e.g., reduced granulosa cell steroidogenesis\nand disrupted estradiol-to-progesterone ratio), impaired oocyte maturation,\novulatory dysfunction, depletion of ovarian reserve, and abnormal\nhyperplasia of the endometrial epithelium. These events may culminate\nin clinical conditions such as POI and endometrial cancer, resulting\nin diminished reproduction capacity. A summary of the MIEs, KEs, and\nAOs discussed is provided in  Table  \n . The KERs were assessed using Bradford-Hill criteria\nand OECD guidelines, \n , \n  as shown in  Table  \n . All of the studies included\nin the assumed AOP framework construction in this article were based\non mammalian models or mammalian/human-derived cell lines. Due to\ninsufficient experimental data from mammalian studies, while two categories\nof nonmammalian models are cited in  Sections  \n  and  3.3  for\nreference, these data were not included in our actual AOP development\nprocess.\nThis study systematically organizes and constructs\nan AOP network\nof GLY-induced female reproductive toxicity ( Figure  \n ), providing a scientific basis for establishing\na health risk assessment system based on early KEs. It also lays a\ntheoretical foundation for the development of prevention and control\nstrategies and the identification of potential intervention targets.\nHowever, several limitations remain: (1) limited research has been\nconducted on the biological mechanisms of GLY and its alternative\nherbicides, as well as the human exposure-relevant doses that can\nbe used for risk characterization. The hazard identification of GLY\nis mostly based on high-dose experiments, while risk characterization\nshould rely on actual human-relevant exposure doses. Many existing\ntoxicological  in vivo  studies use doses several orders\nof magnitude higher than human-relevant exposure levels, lacking  in vivo  studies of low-dose GLY exposure, which may lead\nto misjudgment of the effects of chronic low-dose exposure and make\nit impossible to characterize and assess the actual exposure risks.\n(2) The current evidence supporting the AOP mainly comes from animal\nand in  vitro  experiments, while epidemiological studies\ndirectly linking GLY exposure to human endometrial cancer, POI, and\nfertility decline remain scarce. The available data are insufficient\nto adequately translate experimental findings into human health risk\nassessments. There is an urgent need for more population-based studies\n(e.g., long-term cohort follow-ups) to elucidate the specific effects\nof GLY on female reproductive health. (3) Due to the lack of a recognized\nstandardized conversion method across species/exposure conditions,\nheterogeneous data (e.g., interspecies differences,  in vivo  vs  in vitro  variations, exposure condition discrepancies,\netc.) have not been formally weighted. (4) While qualitative AOPs\ncan effectively integrate mechanistic evidence and identify hazards,\nthey lack the predictive ability required for health risk assessment.\nFor example, the current model lacks quantitative information on response-response\nrelationships, cannot determine the dose threshold for AOs (such as\nPOI onset), and cannot account for population differences in susceptibility.  In contrast, quantitative AOPs (qAOPs) can\naddress these limitations through computational models (such as Bayesian\nnetworks, physiologically based pharmacokinetic models), but their\ndevelopment faces challenges including high data requirements and\nmodeling complexity.  The qualitative\napproach was chosen for this study due to the current insufficient\ndose–response data for GLY across biological levels and the\nparametrization difficulties caused by interstudy heterogeneity (e.g.,\nspecies differences, exposure protocols). The qualitative AOP framework\nprovides a rapidly available conceptual basis under resource-limited\nconditions, although future upgrades to qAOPs will be necessary to\nsupport precise risk assessment.\nAdverse outcome pathway (AOP) network\nrelated to GLY/GBHs-associated\nfemale reproductive toxicity. All KEs, except “compromised\nendometrial receptivity”, are documented in the AOP-wiki and\nare identified by the respective KE IDs. Descriptions of some KE may\ndiffer from those in the AOP-wiki. Color coding: Green represents\nmolecular initiating events; blue represents key events involved in\nmolecular or cellular levels; yellow represents key events involved\nin tissue or organic levels; pink represents adverse outcomes. Solid\nlines indicate adjacent or strongly supported evidence relationships;\ndashed lines indicate nonadjacent or weaker evidence supporting relationship.\nFuture research should focus on: (1) In-depth elucidation\nof the\nbiological mechanisms of GLY and alternative herbicides toxicity,\nparticularly their effects on epigenetic regulation and mitochondrial\nfunction, using human-relevant exposure concentrations to bridge hazard\nidentification and risk characterization. (2) Large-scale epidemiological\nstudies to corroborate findings from experimental models and assess\nreal-world exposure scenarios. (3) To advance the development of quantitative\nAOPs (qAOPs), it is essential to systematically integrate  in vivo  dose–response data from mammalian studies\nto establish computable data sets while developing more systematic\nstandardized approaches including the formulation of species extrapolation\nfactors and unified dose measurement metrics. Priority should be given\nto validating high-impact KERs through  in vitro  experiments\nor  in silico  simulations. Furthermore, quantitative\nanalysis of the associations between mechanistic events and AO probabilities\nusing methods such as benchmark dose (BMD) modeling is crucial for\nsupporting model development. Integrating physiologically based pharmacokinetic\n(PBPK) models with epidemiological data to calibrate qAOP parameters\nwill ultimately enable the comprehensive enhancement of predictive\naccuracy. Addressing these research priorities will provide critical\ntheoretical support for refining the health risk evaluation framework\nof GLY and formulating precise environmental management strategies.","source_license":"CC0","license_restricted":false}