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Relationship: 3746

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Plasma E2, increased leads to Increased, uterine weight

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Plasma E2, increased

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increased, uterine weight

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Activation, estrogen receptor alpha leads to increased uterine weight via earlier proliferation of cells of the uterine lining	adjacent	High		John Frisch (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
mammals	mammals	Moderate	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Female	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Adult, reproductively mature	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Estradiol (E2) is a key signalling estrogen hormone in the hypothalamic–pituitary-gonadal (HPG) axis in the estrus cycle of female rodents and some other vertebrates.

The estrus cycle is a coordinated series of changes that results in fertility in rodents through hormone signaling, including Progesterone, Estradiol, Luteinizing Hormone, and Follicle-Stimulating Hormone, in order to progress through metestrus, diestrus, proestrus, and estrous phases over a period of 4-5 days in rodents, inducing changes in changes to the uterus and vagina (for review see Miller and Takahashi 2014; Swift et al. 2024). In proestrus, increased estradiol levels occur, and physiological changes include ovarian follicle development and the thickening of the uterine wall in preparation for potential pregnancy. Thickening of the uterine wall is caused by proliferation of cells, particularly endometrial epithelial (lining) cells (Nicklaus et al. 2007), causing an increase in uterine weight. In estrus, a surge in luteinizing hormone levels occur, and ovulation of the mature egg. Metestrus is a short transition between estrus and diestrus, features an increase in progesterone levels, and development of the corpus luteum begins in preparation for pregnancy. Diestrus includes continued high levels of progesterone and further development of the corpus luteum; if pregnancy does not occur the corpus luteum regresses and resetting of the cycle occurs.

Increased uterine weight shows that reproductive maturity has occurred because of the association with phases of the estrus cycle, and as an indicator of an adverse outcome when puberty occurs at an earlier or later age than in normal development. Increased estradiol leads to an adverse outcome of puberty occurring at an earlier age than in normal development.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

This Key Event Relationship was part of an Environmental Protection Agency effort to develop AOPs that establish scientifically supported causal linkages between alternative endpoints measured using new approach methodologies (NAMs) and guideline apical endpoints measured in Tier 1 and Tier 2 test guidelines (U.S. EPA, 2025) employed by the Endocrine Disruptor Screening Program (EDSP). A series of key events that represent significant, measurable, milestones connecting molecular initiation to apical endpoints indicative of adversity were identified based on scientific review articles and empirical studies. Additionally, scientific evidence supporting the causal relationships between each pair of key events was assembled and evaluated. The present effort focused primarily on empirical studies with laboratory rodents and other mammals.

Empirical studies are focused on increased plasma estradiol and resulting prolonged estrus cycle, in support of development of AOP 637. Authors of KER 3746 did a further evaluation of published peer-reviewed literature to provide additional evidence in support of the key event relationship. The literature used to support this KER began with the test guidelines and followed to primary, secondary, and/or tertiary works concerning the relevant underlying biology. In addition, search engines were used to target journal articles with term ‘Estradiol’ and ‘Increased uterine weight’ to locate representative empirical studies that support the key event relationship.

Following initial human effort in AOP development, artificial Intelligence (AI)-assisted literature search and synthesis, using EPA-AI GPT5, was used to identify additional literature, and draft Uncertainties and Inconsistencies content of this KER page. Additionally, EPA-AI GPT5 was also used to check for additional text improvement in other sections. All content generated through this process were reviewed and verified by the KER author against literature sources, prior to inclusion.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Increased plasma estradiol leading to increased uterine weight is an established mechanism for onset of estrus cycling from reproductive maturation. Increased plasma estradiol and the timing of increased uterine weight as an adverse outcome have been studied in laboratory mammals by addition of various forms of estradiol (e.g. 17beta-estradiol, ethynyl estradiol; Laws et al. 2000) and toxicants (Li et al. 2008; Bo et al. 2022; Zhou et al. 2023). Studies involving doses of laboratory mammals with various forms of estradiol (e.g. 17beta-estradiol, ethynyl estradiol) are supportive of the mechanism of increases in exposure to estradiol compounds causing increased uterine weight at an earlier age.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Species	Duration	Dose	Increased plasma estradiol?	Increased uterine weight?	Summary	Citation
Rats (Rattus norvegicus)	GD 0 to PND 56	1, 5 mg/kg bw cadmium chloride	yes	yes	Female rats exposed to cadmium chloride had offspring with statistically increased plasma estradiol leading to statistically significant increased uterine weight/body weight ratios at all doses.	Li et al. (2018)
Rats (Rattus norvegicus)	3 days	0.005 mg/kg bw 17-β-estradiol for 3 days or 0.01,0.1 mg/kg bw ethynyl estradiol for 3 days	yes	yes	Female rats exposed to estrogen compounds led to statistically significant increased uterine weight at all doses for both compounds.	Laws et al. (2000)
Mice (Mus muculus)	14 days	High fat diet	yes	yes	Female mice fed high fat diet had statistically significant increased plasma estradiol leading to statistically significant increased uterine weight.	Bo et al. (2022)
Mice (Mus muculus)	20 days	2 ug/g bw leptin daily	yes	yes	Female mice injected with leptin had statistically significant increased plasma estradiol leading to significant increased uterine weight.	Zhou et al. (2023)

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Temporal alignment and biphasic uterine response
- In laboratory rodents, the uterus exhibits an early increase in weight from water imbibition followed by growth, so the observed magnitude depends strongly on necropsy timing (OECD 2007).
- In laboratory mice, genomic and histological data also show biphasic early/late uterine responses to estradiol over 0.5–24 h, reinforcing that sampling windows can capture different biological events, with initial response within the first hour including water imbibition, with subsequent response up to 24 hours later including development of tissue layers (Hewitt et al. 2005).
- Empirically, some estrogens statistically increase uterine weight in laboratory rats at 6 hours but not 24 hours after the last dose, creating apparent inconsistencies based on when necropsy occurs (Laws et al. 2000).
Route of exposure and kinetic differences
- Method of administered dose (i.e. subcutaneous vs. oral routes) can yield different magnitude of uterotrophic responses in laboratory rats, with greater uterine weight gain in animals exposed via subcutaneous injection than those animals exposed via oral gavage in Laws et al. (2000) for 4-tert-octylphenol (200-400 mg/kg body weight) and bisphenol A (200 mg/kg body weight), but greater uterine weight gain in animals exposed via oral gavage than those animals exposed via subcutanious injection for methoxychlor (50 mg/kg body weight) or 4-nonylphenol (50-200 mg/kg body weight) in Laws et al. (2000).
Strain effects
- Some laboratory rodent strains are less responsive to administered doses. OECD (2007) recommends use of commonly used laboratory rodent strains (i.e. Sprague-Dawley and Wistar strains of rats) with demonstrated uterotrophic responses.
Cross-talk and hormone confounding effects.
- Estrogen receptor alpha is a nuclear receptor, and cross-talk signalling to other nuclear receptors can trigger additional gene expression pathways and mechanisms. Reproductive maturation is influenced by a variety of hormones (i.e. progesterone), with uterine weight gain not exclusively caused by increased estradiol in laboratory mice (Hewitt et al. 2025).
Effects of phytoestrogens
- A low phytoestrogen diet is recommended, as high levels of phytoestrogens in diet have been shown to have confounding effects in empirical studies of laboratory rodents due to estrogenic effects (OECD 2007).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Life Stage: Applies to adult, reproductively mature.

Sex: Applies to females as specific to uterus.

Taxonomic: Primarily studied in laboratory rodents. Plausible for mammals and marsupials that have uteri.

References

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

Bo T, Liu M, Tang L, Lv J, Wen J, Wang D. 2022. Effects of High-Fat Diet During Childhood on Precocious Puberty and Gut Microbiota in Mice. Frontiers in Microbiology 13: 930747.

Hewitt SC, Harrell JC, Korach KS. Lessons in estrogen biology from knockout and transgenic animals. Annu Rev Physiol. 2005;67:285-308.

Laws SC, Carey SA, Ferrell JM, Bodman GJ, Cooper RL. 2000. Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. Toxicological Sciences 54(1):154-167.

Li Z, Li T, Leng Y, Chen S, Liu Q, Feng J, Chen H, Huang Y, Zhang Q. 2018. Hormonal changes and folliculogenesis in female offspring of rats exposed to cadmium during gestation and lactation. Environmental Pollution 238: 336-347.

Miller, B.H. and Takahashi, J.S. 2014. Central circadian control of female reproductive function. Frontiers in Endocrinology 4(1): 195.

Niklaus AL, Aubuchon M, Zapantis G, Li P, Qian H, Isaac B, Kim MY, Adel G, Pollard JW, Santoro NF. 2007. Assessment of the proliferative status of epithelial cell types in the endometrium of young and menopausal transition women. Human Reproduction 22(6): 1778-1788.

Organisation for Economic Co-operation and Development. 2007. Test No. 440: Uterotrophic Bioassay in Rodents: A short-term screening test for oestrogenic properties, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. https://ntp.niehs.nih.gov/sites/default/files/iccvam/suppdocs/feddocs/oecd/oecdtg440.pdf (retrieved 6 May2026).

Swift, K.M., Gary, N.C., and Urbanczyk, P.J. 2024. On the basis of sex and sleep: the influence of the estrous cycle and sex on sleep-wake behavior. Frontiers in Neuroscience 18:1426189.

U.S. Environmental Protection Agency. 2025. EDSP Test Guidelines and Guidance Document. https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/edsp-test-guidelines-and-guidance-document (retrieved 25 July 2025).

Zhou L, Ren Y, Li D, Zhou W, Li C, Wang Q, Yang X. 2023. Timosaponin AIII attenuates precocious puberty in mice through downregulating the hypothalamic-pituitary-gonadal axis. Acta Biochimica Polonica 70(1): 183-190.

Italics indicate edits from John Frisch March 2026. A full list of updates can be found in the Change Log on the View History page.