AOP: 563

Title

A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Aryl hydrocarbon Receptor (AhR) activation causes Premature Ovarian Insufficiency leading to Reproductive Failure

Short name

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AhR activation leading to Premature ovarian insufficiency

The current version of the Developer's Handbook will be automatically populated into the Handbook Version field when a new AOP page is created.Authors have the option to switch to a newer (but not older) Handbook version any time thereafter. More help

Handbook Version v2.7

Graphical Representation

A graphical representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help

Click to download graphical representation template Explore AOP in a Third Party Tool

Authors

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Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section. More help

Sapana Kushwaha (email point of contact)

Contributors

Users with write access to the AOP page. Entries in this field are controlled by the Point of Contact. More help

Sapana Kushwaha
Prajakta Ghume
Mahesh Rachamalla

Coaches

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Dan Villeneuve

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication. More help

OECD Project #	OECD Status	Reviewer's Reports	Journal-format Article	OECD iLibrary Published Version

This AOP was last modified on February 14, 2025 05:08

Revision dates for related pages

Page	Revision Date/Time
Activation, AhR	February 28, 2024 05:12
dimerization, AHR/ARNT	January 25, 2025 05:32
increased, Bax expression	January 26, 2025 05:47
Apoptosis	February 28, 2024 09:40
Decreased, size of the ovarian reserve	December 17, 2024 15:22
Premature ovarian Insufficiency	January 23, 2025 05:43
Activation, AhR leads to dimerization, AHR/ARNT	March 22, 2018 11:02
Activation, AhR leads to Decreased, ovarian reserve	January 22, 2025 04:13
dimerization, AHR/ARNT leads to increased, Bax	February 13, 2025 02:06
increased, Bax leads to Apoptosis	February 15, 2025 07:04
Apoptosis leads to Decreased, ovarian reserve	January 28, 2025 07:04
Decreased, ovarian reserve leads to POI	January 28, 2025 02:15
Polycyclic aromatic hydrocarbons (PAHs)	February 09, 2017 15:43
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)	February 09, 2017 14:32
7,12-Dimethylbenz(a)anthracene	January 06, 2025 01:21
benzo[a]pyrene	October 30, 2019 16:47

Abstract

A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

Premature ovarian insufficiency (POI) is marked by a decline in ovarian reserve and reduced production of sex hormones, often leading to early menopause. Environmental toxicants are increasingly recognized as major factors influencing ovarian health, with effects evident both during prenatal development and adulthood. Many of these substances exert their impact by activating the aryl hydrocarbon receptor (AhR), a nuclear transcription factor involved in metabolizing xenobiotics and regulating ovarian functions.

AhR influences crucial reproductive processes, such as estrogen synthesis, follicle maturation, and ovulation. When activated by environmental contaminants—including polyaromatic hydrocarbons, dioxins (e.g., TCDD), endocrine disruptors, flame retardants, and diesel particulates—AhR triggers the expression of pro-apoptotic genes like bax. This activation leads to oocyte apoptosis, ultimately depleting the ovarian follicle pool and disrupting hormonal balance and reproductive processes. Prolonged exposure to such toxicants accelerates ovarian aging and heightens the risk of POI, significantly affecting fertility in humans and animals.

Research in animal models and human studies has shown that AhR activation impairs follicle development and diminishes ovarian reserve through apoptotic pathways. This mechanistic understanding forms the basis of an adverse outcome pathway (AOP) that links toxicant-induced AhR activation to POI. This AOP framework can support risk assessments and inform strategies to mitigate the reproductive health risks associated with environmental toxicant exposure.

AOP Development Strategy

Context

Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

The female reproductive system includes the ovaries, fallopian tubes (or oviducts), uterus, and vagina, and is hormonally regulated by the hypothalamus and pituitary gland. All these organs function in conjunction to fulfil the main roles of the female reproductive system (1). The process that describes the growth and development of follicles, or atresia, through a sequence of morphological and functional phases from primordial to ovulatory is known as folliculogenesis (2). Oogenesis, the process of creating new primordial germ cells within the epiblast, starts in the human ovary at 2 weeks post-conception (wpc). The cells then move to the hind intestine, settle in the gonadal ridges before 7 wpc, and multiply into ovarian cysts, also known as nests, as oogonia. Primordial granulosa cells, which originate from the gonadal ridges, are also found in the cysts (3). The oogonia stops mitosis, expands, and mature into primary oocytes after 8.5 wpc, starting the meiotic prophase. The disintegration of the cysts, which takes place between 13 and 30 wpc, results in the development of 30-mm-diameter primordial follicles. These follicles are made up of a monolayer of squamous granulosa cells encircling a primary oocyte that has been arrested during the diplotene stage of prophase I of meiosis (4). According to theories, the newly produced primordial follicles serve as the female's limited supply of resting oocytes for the duration of her reproductive life (5). The number of primordial follicles formed within the ovary throughout development is the result of the widespread proliferation and death of primordial germ cells, oogonia, and oocytes (6). Females are born with an average of 400,000-700,000 primordial follicles, which diminish considerably over the reproductive lifespan (7).Follicles can be classified into three types:

Nongrowing, primordial follicles are described as isolated oocytes or oocytes surrounded by a partial or intact single layer of granulosa cells.
Growing follicles are described as oocytes surrounded by a multi-layered, solid covering of granulosa cells with no sign of a fluid-filled cavity (antrum).
Antral follicles consist of a core oocyte and a fluid-filled antrum surrounded by hundreds of stratified granulosa cells (8).

POI is a medical condition in which ovarian follicles become exhausted and no longer function normally as reproductive and endocrine organs in women under the age of 40. It is characterized by low ovarian sex hormone levels and a reduced number of ovarian follicles, hastening the onset of menopause. This condition frequently results in subfertility or infertility due to hypoestrogenism, which causes menstrual abnormalities and pregnancy failures. Approximately 1% of women under the age of 40 experience POI (9). Four primary mechanisms can result in POI:

Depletion of the reserve of dormant primordial follicles, either due to inherent factors leading to their failure in assembly or acquired reasons causing increased atresia.
Increased occurrence of follicular atresia.
Pronounced activation of primordial follicles.
Interruption of follicular development before reaching the antral stages, thereby hindering ovulation (10).

Follicle depletion means that there are no primordial follicles left in the ovary. This condition can be caused by several factors, including failure to produce an adequate initial pool of primordial follicles during gestation, rapid follicle turnover, or autoimmune or toxic destruction of follicles (11).

The Aryl Hydrocarbon Receptor (AHR) features a Per-ARNT-Sim (PAS) domain that binds to a range of endogenous and exogenous substances. It is a member of the basic helix-loop-helix (bHLH) family. In its unliganded form, AHR is part of a cytosolic complex containing heat shock protein 90 (HSP90), the HSP90 co-chaperone p23, and the AHR-interacting protein (AIP). Upon ligand binding, AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT). The AHR-ARNT complex then binds to recognition sequences in the DNA, known as AHR response elements (AhREs) or xenobiotic response elements (XREs), which contain the core motif 5’-GCGTG-3’. These interactions lead to the induction or repression of gene expression (Fig.2) (12). AHR controls the rate at which oocytes in germ cell nests undergo apoptosis during the embryonic stage, as well as the survival of oocytes in the ovaries of the foetus and newborn. Ligands of the AHR are classified into two categories: (i) anthropogenic compounds, which include aromatic environmental contaminants and various pharmaceuticals, and (ii) natural compounds, which are produced by bacteria, plants, animals, or the human body itself (13). Environmental factors known to activate AHR appear to be significant triggers of reduced ovarian reserve or early menopause, affecting both the prenatal period and adult life. These factors are believed to play a role in the development of POI (14). Apoptosis is the organized process of cell death characterized by membrane blebbing, cell shrinkage, chromatin condensation, and DNA fragmentation, followed by the rapid engulfment of the cell remnants by neighboring cells. Often referred to as "physiological cell death," apoptosis plays a crucial role in cell turnover, physiological involution, and the atrophy of various tissues and organs (15). The Bcl-2 protein family inhibits apoptosis by binding to and blocking pro-apoptotic proteins, such as Bax and Bak, preventing their translocation to the mitochondrial outer membrane. In the absence of Bcl-2 inhibition, Bax and Bak induce damage to the mitochondrial membrane, leading to the release of pro-apoptotic signaling proteins like cytochrome c. This release triggers the activation of the caspase cascade, which ultimately promotes apoptosis (Fig.2). When environmental contaminants activate the AhR, it leads to an increased production of pro-apoptotic proteins. This, in turn, causes apoptosis in ovarian follicles, resulting in follicular pool depletion and a reduced ovarian reserve (Fig.2) (16). Numerous studies have demonstrated that the biological effects of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and other potent AhR ligands are consistent with the activation of AhR (17, 18). These studies have also shown that exposure to AhR ligand increases the expression of apoptotic genes, the production of Bax protein, and the subsequent Bax-dependent increase in apoptosis in murine oocytes. These findings highlight the roles that AhR signalling and apoptosis play in delayed follicle development, survival, and oocyte death (19).

Environmental factors influencing pregnancy or adulthood are significant predictors of ovarian reserve and early menopause. A cross-sectional study using data from the National Health and Nutrition Examination Survey (NHANES) involving 31,575 women from 1999 to 2008 found that, after adjusting for age, race/ethnicity, smoking, and body mass index, nine polychlorinated biphenyls (PCBs), three pesticides, one furan, and two phthalates among 111 endocrine-disrupting chemicals (EDCs) were significantly associated with an earlier onset of menopause, ranging from 1.9 to 3.8 years (10). Premature ovarian pool exhaustion, a primary characteristic of primary ovarian insufficiency (POI), was attributed to the combination of two common polycyclic aromatic hydrocarbons (PAHs), 7,12-dimethylbenz(a)anthracene and benzo[a]pyrene (BaP). This combination exposed fetuses to the mixture at an accelerated rate (20). Most of these environmental pollutants affect the ovaries through the aryl hydrocarbon receptor (AhR), leading to follicular apoptosis at various stages. This results in a reduced number of follicles available for fertilization or menstruation, contributing to issues such as early menopause and infertility due to diminished ovarian reserve and exacerbating POI.

Strategy

Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components. Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

The AOP is developed and updated following the framework outlined in the OECD Adverse Outcome Pathway (AOP) Handbook.

1. Literature Review and Data Extraction

The pathway is identified and validated through ongoing searches in prominent research databases such as PubMed, Google Scholar, ScienceDirect, semantic scholar, Researchgate, SpringerLink and Elsevier. This ensures continuous incorporation of relevant evidence.

Keywords Used: AHR, ARNT, bax, Premature Ovarian Insufficiency (POI), Premature Ovarian Failure (POF), female reproductive system, fertility, polyaromatic hydrocarbons, environmental toxicants, folliculogenesis, apoptosis in ovary.

2. Identifying and Structuring Key Events (KEs)

Key Events (KEs) are identified and organized based on their roles in the pathway from the MIE to the AO. Each KE is evaluated for its essentiality and relevance in the pathway. KEs are clearly outlined as critical biological processes that follow the MIE and culminate in the AO. These events are selected to reflect the mechanistic progression and functional changes essential to the pathway.

3. Biological Plausibility

Each KE is evaluated for biological plausibility by demonstrating the biological relevance of each KE with reference to molecular interactions, cellular processes, and their roles in the progression of the pathway. Citing peer-reviewed literature, experimental data, and established biological principles that validate the occurrence and role of each KE.

4. Empirical Evidence

Empirical evidence supporting each KE synthesized from experimental studies, results from in vitro studies, animal models, and human data showing the activation of each KE in response to the MIE. Evidence linking exposure to environmental toxicants or stressors with measurable effects on each KE. Studies demonstrating the occurrence of KEs in conditions relevant to the AOP, including POI/POF, folliculogenesis impairment, and apoptosis in ovarian tissues.

5. Temporal Relationships

The sequence of events in the pathway is delineated to establish a logical progression. Each KE is placed in its appropriate temporal context, illustrating how earlier events influence downstream processes. Experimental and observational studies are used to confirm causal evidence that one KE precedes and leads to the activation of the next.

6. Concordance Across Systems

To strengthen the confidence in the AOP, concordance across different systems is assessed. Evaluating whether KEs are conserved across species or model systems. Examining the reproducibility of KEs under various experimental and environmental conditions to validate their relevance.

Summary of the AOP

This section is for information that describes the overall AOP.The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)

An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help

Key Events (KE)

A measurable event within a specific biological level of organisation. More help

Adverse Outcomes (AO)

An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help

Type	Event ID	Title	Short name

MIE

Activation, AhR

KE	944	dimerization, AHR/ARNT	dimerization, AHR/ARNT
KE	2124	increased, Bax expression	increased, Bax
KE	1262	Apoptosis	Apoptosis
KE	1883	Decreased, size of the ovarian reserve	Decreased, ovarian reserve

2305

Premature ovarian Insufficiency

POI

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Title	Adjacency	Evidence	Quantitative Understanding

Activation, AhR leads to dimerization, AHR/ARNT	adjacent	High
dimerization, AHR/ARNT leads to increased, Bax	adjacent	Moderate
increased, Bax leads to Apoptosis	adjacent	High
Apoptosis leads to Decreased, ovarian reserve	adjacent	Moderate
Decreased, ovarian reserve leads to POI	adjacent	High

Activation, AhR leads to Decreased, ovarian reserve

non-adjacent

Moderate

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Name
Polycyclic aromatic hydrocarbons (PAHs)
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
7,12-Dimethylbenz(a)anthracene
benzo[a]pyrene

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help

Life stage	Evidence
Fetal	High
Adult	Moderate

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help

Term	Scientific Term	Evidence	Link
rat	Rattus norvegicus	High	NCBI
mouse	Mus musculus	High	NCBI
human	Homo sapiens	Moderate	NCBI
zebra fish	Danio rerio	Low	NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help

Sex	Evidence
Female	High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

This Adverse Outcome Pathway (AOP) is pertinent to both early-life and adult female exposure to various chemicals, including environmental contaminants, which can result in reproductive failure (1, 57-60). The experimental evidence supporting this pathway is derived from studies conducted on rodent models, as well as human mechanistic and epidemiological data. Data from experimental animal studies are considered relevant and applicable for assessing human risk.

Essentiality of the Key Events

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

Event ID	Key Event	Evidence	Essentiality Assessment
18	Activation, AhR	Strong	Numerous research conducted both in vitro and in vivo using model organisms such as rodents has shown that animals lacking the AhR gene in knockout studies retain a significantly larger primordial follicle pool than controls and have either reduced or no harmful effects of PAHs, DMBA, etc. on follicles. Over a 24-hour period, the ovaries of newborns with AhR deficiency were unable to increase the expression of the Hrk mRNA in response to PAH (21). AhR is necessary for the PAH-induced rupturing of oocytes. In contrast to the toxic responses observed in wildtype (WT) ovaries following exposure to 0.1 μM DMBA-DHD, the ovaries of AhR deficient mice do not exhibit heightened Bax production (24 hours post-PAH exposure) nor oocyte damage (48 hours post-PAH exposure) after being treated with 0.1 μM DMBA-DHD (22).
944	Dimerization, AHR/ARNT	Strong	In canonical AhR signalling, the cytoplasmic inactive form of AhR is changed into its active form, which may dimerise with the AhR nuclear translocator (ARNT) and enter the nucleus. There is proof to back the notion that the AHR/ARNT heterodimer can control gene expression within the Ahr signalling cascade (13, 23)
2124	Increased, Bax expression	Moderate	1. Bax is functionally necessary for the destruction of oocytes caused by PAHs, as animals lacking Bax maintain normal oocyte counts even after being exposed to PAHs. According to research on foetal ovarian germ cells, Bax is a crucial modulator of AHR-driven oocyte death. The ovarian reserves of Bax-deficient female foetuses revealed to PAHs concurrently had a normal count of primordial follicles at birth suggests that functional Bax protein is essential for the fetal ovarian harm resulting from transplacental exposure to PAHs (24). In neonatal ovaries, deletion of the apoptotic inducing protein BAX results in more primordial follicles and oocytes than the Wild type (control) (25). Analysis of animals with a particular bax gene deficiency has validated the the function of bax in the apoptosis of ovarian cells, as indicated by findings from the investigation of variations in bax mRNA levels. Granulosa cells' capacity to undergo apoptosis appeared to be impaired in ovaries of BAX-deficient animals, according to histologic examination (26). Excess follicles were seen in aged (1-year-old) female Bax KO animals at nearly every developmental stage, aggravating the phenotype brought on by Bax single KO (27). In research on three types of adult female characiform fish, the immunostaining of the ovaries for bax was described as patches scattered all over the cytoplasm. In the latter stages of follicular atresia, the cytoplasm of follicular cells, granulocytes, and thecal cells showed the labelling for bax (28). In human foetal ovaries, the apoptosis-inducing protein BAX displayed distinct signals associated with oogonia, oocytes contained in primordial follicles, and pregranulosa and granulosa cells (29). In rats, low levels of 3-methylcholanthrene (3MC) during the pubertal stage interfere with follicular development and ovulation through AhR binding to gene promoter areas related to Bax-mediated apoptosis (30). DMBA-treated ovaries from PND4 Fisher 344 female rats indicate that follicular loss brought on by DMBA is linked to elevated pro-apoptotic Bax expression at the time of follicle depletion (day 8), demonstrating that Bax activation occurs concurrently with follicular destruction and is a component of the apoptotic response that drives ovotoxicity (31). Research conducted on F1 hybrid mice (C57BL/6j × CBA/Ca) has shown that B[a]P exposure triggers the AhR signaling pathway, which results in the dysregulation of AhR signaling-related gene expression. This activation also significantly increases Bax expression, which is involved in the pathway of cell death.These findings imply that both the AhR signaling pathway and the cell death mechanisms may be crucial factors contributing to impaired follicle development (32).
1262	Apoptosis	Strong	1. In addition to being mentioned in the AOP-Wiki KE 1262 implicated in related AOPs, increased bax expression has been shown to be a pro-apoptotic factor that causes apoptosis in published literatures (15, 33). Research on the regulation of apoptosis indicates that animals lacking the Bax gene have an abundance of follicles and suppression of ovarian apoptosis (34). Granulosa cells found in the ovarian follicles of BAX-deficient animals appear to be immune to the activation of apoptosis during atresia. Granulosa cells from early atretic follicles in the human ovary have significant expression of BAX, despite the fact that BAX is either minimal or undetectable levels are observed in both healthy and significantly atretic follicles (26). Research on the ovaries of quail shows that BAX is essential for apoptosis, especially when follicular atresia occurs. BAX immunoreactivity (IR) was detected in the nuclei of some prelampbrush oocytes and within the follicular cells of atretic follicles,indicating that it is involved in granulosa cell and oocyte death. In fully degraded atretic follicles, BAX was not present, suggesting that its function as a pro-apoptotic regulator is limited to the early phases of apoptosis (35). Vinclozolin (VCD)-exposed female mice showed that Bax is essential for follicular apoptosis in both the primordial and primary pools, potentially functioning upstream in the VCD-induced apoptotic pathway. Significant defence against caused by VCD (vinclozolin)destruction of both primordial and primary follicles was demonstrated by Bax-null animals (36).
1883	Reduced, Ovarian reserve (follicle pool)	Moderate	1. Apoptosis brought on by ligand activation of AhR and the reduction of the folicle pool resulting from the above processes of bax expression have been demonstrated in multiple studies to be a downstream reaction to AhR activation in ovaries. An investigation into cumulus-oocyte complexes (COCs) showed exogenous AhR ligands decrease oocyte maturation and cause apoptosis in cumulus cells. may contribute to follicular apoptosis by upregulating Bax expression. This demonstrates how AhR, Bax, and apoptosis interact to cause follicular loss and ovotoxicity (37). Morphological analysis of follicles at numerous stages of development, including multiple big oestrogenic antral follicles with intact oocytes, were seen in the ovaries of old female Bax−/−mice. This contrasts with wild-type mice, which exhibit a diminished follicle pool. The ovarian structure in Bax−/− mice resembles that of young adult females and slows the depletion of primordial and primary follicles postnatally by conferring upon granulosa cells and oocytes a degree of resistance to apoptosis in the context of ovarian maturation (38). According to a study, PAH-activated AHR causes foetal ovarian germ cells to undergo apoptosis by upregulating Bax expression, which results in a decrease in ovarian follicle reserves. AHR and Bax are prominent mediators of germ cell loss, and their roles in toxicant-induced ovarian damage are suggested by the fact that Bax deficiency inhibits this apoptosis, maintaining follicle counts despite PAH exposure (24). DMBA-induced apoptosis using gene-mutant mice that results in follicle loss and oocyte depletion due to PAH exposure (39). By upregulating BAX expression and activating AHR, maternal exposure to PAHs causes foetal germ cell apoptosis and induces apoptosis of chicken primordial germ cells (PGC). Using RNA interference and antagonist technology to suppress AHR expression, it was discovered that AHR silencing could fully reverse the negative effects of PAH on germ cell differentiation, demonstrating the functional necessity of AHR in this process (40).
AO	Premature Ovarian Insufficiency	-	Research on how polycyclic aromatic hydrocarbons (PAHs), like DMBA, cause oocyte apoptosis in mouse and human ovarian models by upregulating the pro-apoptotic gene Bax through AhR, which results in oocyte destruction and premature ovarian failure (22). According to a number of studies, reduced ovarian reserve (DOR) is the term used to describe a scarcity of follicles at birth or an abrupt reduction in follicle counts at birth or following adolescence. Primary ovarian insufficiency may result from DOR (11, 41). A study involving 157 POF patients examines the link between women's premature ovarian failure (POF) and being subjected to (PAHs). Elevated concentrations of (PAHs) have been linked to a reduction in ovarian reserve., hormonal abnormalities (elevated FSH and LH, decreased AMH), and increased chances of POF. The study emphasises how aryl hydrocarbon receptor (AhR) signalling is probably the mechanism by which PAHs have a harmful impact on ovarian function, resulting in follicle depletion and compromised reproductive health (20, 42). Research studies comparing consequences of prolonged exposure to environmental contaminants, such as dioxins and heavy metals, on ovarian function in women from contaminated areas (Taranto, Italy) to those from non-polluted areas. In the granulosa cells of Taranto women, the results reveal elevated expression of AhR and its cofactor ARNT, which results in changed gene expression and regulation of apoptosis. This links AHR activation to ovarian failure by contributing to decreased ovarian function, which is shown by abnormal hormonal profiles and fewer mature oocytes (43).

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

Biological Plausibilty

AhR activation and ARNT dimerization: Strong- There is strong evidence of biological plausibility from other well-established AOPs on Ahr activation. Many studies have been conducted on the functions of Ahr and its binding partners, such as ARNT (44), and it is widely recognised that a number of physiological and toxicological processes are mediated by the Ahr signalling pathway (45). Studies showing AhR controls the quantity of pre-antral and antral follicles provide strong evidence for the AhR's involvement in controlling ovarian follicle development in latter phases of folliculogenesis (1). The canonical evidence for AhR signalling is established in a number of publications. Numerous exogenous substances, particularly TCDD, PAH, EDCs, and others, are recognised to attach to and activate the AhR, causing harmful reactions in the ovaries. It should be noted that this KER is already included in a number of well-established AOPs with abundance evidence listed on the AOP-wiki. Refer below the citations: (12, 13, 17, 46)

AhR dimerization and bax expression: Moderate- A common category of industrial and ecological biohazards activates (AhR), which further dimerises through a canonical pathways, accelerating up the depletion of germ cells in female mice during pregnancy and the postnatal period. Bax's ovarian expression, a significant proapoptotic gene cassette that operate at several stages of the cell death signalling cascade, is upregulated by AhR-activated PAH (39). Animals lacking Bax show nearly total resistance to the loss of this follicle type, suggesting that the stressor-activated AhR complex's transcriptional control Bax, a member of the pro-apoptotic Bcl-2 family, is crucial for initiating cell death in primordial follicles (21). The findings demonstrate that a single intraperitoneal injection of 9,10-dimethylbenz[a]anthracene (DMBA), a prototypical PAH, dramatically raises ovarian Bax mRNA levels and causes a consistent buildup of Bax protein in quiescent (primordial) and early developing (primary) oocytes. This suggests that the AhR transactivates this crucial proapoptotic gene in oocytes. These findings suggested that the PAH-activated AhR directly regulates Bax since oocytes exhibit both the AhR and the AhR nuclear translocator, which is its dimerization partner (22).

Increased Bax and apoptosis in oocytes: Strong- The pro-apoptotic protein Bax is essential for the ovarian follicles' apoptosis caused by PAHs. PAHs, such as those found in cigarette smoke, cause follicular cell death by activating the AhR pathway and upregulating pro-apoptotic proteins such bax (21). Both the rat and human ovaries' granulosa cells as well as the luteal cells of the bovine ovary, apoptosis is positively connected with elevated expression of Bax, a factor linked to mortality. In neonatal ovaries, deletion of the pro-apoptotic protein Bax results in more oocytes as primordial follicles than WT (25). The cytotoxic response's functional relevance of increased Bax expression is supported by the observation that Bax-deficient female foetuses subjected in parallel did not exhibit foetal loss of ovarian germ cells as a result of wild-type female foetuses being subjected to PAHs during pregnancy. We conclude that the AhR is essential for transmitting PAH's activities in foetal ovarian germ cells, and that a key mediator of PAH-induced oocyte loss in female foetuses who receive PAHs during pregnancy is the proapoptotic Bcl-2 family member Bax. Research has demonstrated that recombinant Bax protein microinjection into oocytes directly causes apoptosis, suggesting that a high concentration of Bax is both necessary and adequate to cause the death of female germ cells (21, 25, 26, 47, 48).

Apoptosis and reduced follicle pool: Moderate- The biological plausibility of this relationship is moderate, as the ovary's oocyte production is limited established at birth. According to studies, adding BaP to culture media causes foetal oocytes to undergo apoptosis and raises the expression of Bax. Reduced ovarian reserve at birth is the result of permanent effects from prenatal exposure to BaP. Similarly, exposure to 4(3H)-quinazolinone-2-ethyl-2-phenyl ethyl (QEPE) has been found to trigger apoptosis in mouse foetal oocytes by a process requiring elevated expression of Bax. Furthermore, female mouse fetuses exposed in utero (PAHs) exhibit a significantly decreased stock of primordial follicles that produce eggs, as demonstrated in extensive rodent studies (24, 49-51). Research on the ovarian tissues of humans and baboons indicates that apoptosis takes place during and is most likely the cause of follicular atresia in baboons and humans ovaries. The bax's immunohistochemistry localisation revealed that it was non-detectable in healthy follicles but abundant in the granulosa cells of early atretic follicles. Antral follicles are reduced in AhR-deficient mice, while germ cells and primordial follicles are increased (30, 52).

Follicle pool reduction and POI: Moderate- Since the relationship between follicle pool reduction and the onset of POI has been shown in a number of rodent species and in some human surveys showing that activation of AhR results in granulosa cells and oocytes being destroyed, which causes the development of premature menopause and ovarian dysfunction, the biological plausibility of this KER is moderate (53). Direct exposure to PAHs or cigarette smoke speeds up the primordial follicles' demise, which causes the ovarian pool to be exhausted too soon and ultimately results in premature ovarian failure. According to well-established research, the majority of PAHs with coplanar structures bind (AhR) to cause harm. The primary feature of POF is early ovarian pool depletion, which is caused by accelerated loss of primordial follicles caused by perinatal exposure to a combination of two prototypical PAHs, 7,12-dimethylbenz(a)anthracene and BaP. Matikainen T. has demonstrated that expression of the Bax gene, which is triggered by the Aryl hydrocarbon receptor, is necessary to prevent premature ovarian failure caused by biohazardous environmental pollutants. In the case of humans, any rise in the follicle depletion rate may increase the risk of early menopause and early sexual senescence, or premature ovarian failure (54). Prenatal or postnatal exposure to diesel exhaust (PAH chemicals) reduces the primordial and primary follicular pool, which results in POF and impairs the female's future reproductive capacity (55, 56).

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help

Modulating Factor (MF)	Influence or Outcome	KER(s) involved

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors. More help

Considerations for Potential Applications of the AOP (optional)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

The AOP describes a pathway that facilitates the identification of aryl hydrocarbon receptor (AHR)-related modes of action induced by contaminants, with a focus on substances that adversely affect the reproductive system. This pathway establishes a robust foundation for linking AHR activation to apical reproductive endpoints, a critical step in identifying endocrine-disrupting chemicals (EDCs). EDCs are subject to specific evaluation under regulations such as REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals; Regulation 1907/2006, EU), the revised European Plant Protection Product Regulation 1107/2009 (EU), and the Biocidal Products Regulation 528/2012 (EU). Furthermore, agencies such as the US EPA have prioritized the study and regulation of EDCs (EPA, 1998).
This AOP represents the current understanding of the pathway from AHR activation to impaired fertility, offering a framework for developing and interpreting Integrated Approaches to Testing and Assessment (IATA). These strategies aim to identify substances with similar mechanisms of action related to AHR-mediated disruption of sex steroid signaling and adverse effects on the reproductive system and fertility. This AOP serves as a foundational element for mapping a broader AOP network addressing modes of action associated with endocrine disruption.
Additionally, the AOP can guide the development of quantitative structure-activity relationships (QSARs), read-across models, and systems biology frameworks to prioritize chemicals for further testing and evaluation.
Risk Assessment: This AOP is valuable for evaluating the risk posed by various environmental toxicants in developing premature ovarian insufficiency (POI).
Drug Targeting: Investigating AhR pathways can aid in identifying potential drug targets.
Linking Ovarian Dysfunction and Fertility: The proposed AOP can help establish connections between ovarian dysfunction and reduced fertility in females.
Mechanisms of Ovarian Toxicity: It will advance understanding of how pollutants/toxicants activating AhR cause ovarian toxicity and highlight the need for further screening of chemicals to address their potential to deplete follicles and progress POI.
Preventive Measures: The study will illustrate the processes behind ovarian toxicity caused by AhR-activating pollutants, emphasizing the need for improved chemical screening to prevent follicle depletion and the advancement of POI.

References

List of the literature that was cited for this AOP. More help

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