This Key Event Relationship is licensed under the Creative Commons BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

Relationship: 3769

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 prolonged, estrus cycle

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

prolonged, estrus cycle

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 decreased fertility via failure to ovulate	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 Do not cite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
Placental Mammals	Eutheria	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
Juvenile	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. 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.

A prolonged estrus period occurs due to a disruption of the estrus cycle.

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, 2024) 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 640.

Authors of KER 3769 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’, ‘Prolonged estrus cycle’, and ‘Persistent estrus cycle’ to locate representative empirical studies that support the key event relationship.

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 and prolonged estrus cycle have been studied in laboratory mammals by addition of various forms of estradiol (e.g. 17beta-estradiol; Kouki et al. 2005; Takahashi et al. 2013; Shirota et al. 2015). Studies involving dosing of laboratory mammals with various forms of estradiol (e.g. 17beta-estradiol) are supportive of the mechanism of increases in exposure to estradiol compounds causing prolonged estrus cycle. Increased estradiol has been shown to disrupt the estrus cycle by altering the hormone levels in rodents, with neonatal estrogenization disrupting signalling in the hypothalamic–pituitary-gonadal (HPG) axis leading to prolonged estrus (Takahashi et al. 2013; Shirota et al. 2015).

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?	Prolonged estrus cycle?	Summary	Citation
Rats (Rattus norvegicus)	60 days	1 mg/0.05 ml oil 17B-estradiol injected day 5.	yes	yes	Rats injected with estradiol led to statistically significant decreased frequency of regular estrus cycle, with increased frequency of prolonged and persistent estrus.	Kouki et al. (2005)
Rats (Rattus norvegicus)	10 months	0.02, 0.2, 2, 20, or 200 ug/kg of body weight 17α-ethynylestradiol injected within 24 hours of birth.	yes	yes	Rats injected with ethynylestradiol had statistically significant increase in the incidence of abnormal estrus cycles in 10-week-old rats at 200 ug/kg, 14-week-old rats at 20 ug/kg, 18-week-old rats at 2 ug/kg and 22-week-old rats at 0.2 ug/kg compared to the 0 ug/kg group by persistent estrus.	Takahashi et al. (2013)
Rats (Rattus norvegicus)	23 weeks	0.4, 2 ug/kg/d oral 17α-ethynylestradiol for 5 days beginning post-natal day 1.	yes	yes	Female mice exposed to ethynylestradiol had increased frequency of persistent estrus and irregular estrus cycles, with statistically significant decreased estrus cycles and statistically significant increased number of estrus/proestrus days at all doses.	Shirota et al. (2015)

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

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 ovaries.

Taxonomic: Persistent estrus is primarily studied in laboratory rodents. Plausible for placental mammals that have an estrus cycle.

References

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

Kouki T, Okamoto M, Wada S, Kishitake M and Yamanouchi K. 2005. Suppressive effect of neonatal treatment with a phytoestrogen, coumestrol, on lordosis and estrous cycle in female rats. Brain Research Bulletin 64: 449-454.

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

Shirota M, Kawashima J, Nakamura T, Kamiie J, Shirota K, Yoshida M. 2015. Dose-dependent acceleration in the delayed effects of neonatal oral exposure to low-dose 17α-ethynylestradiol on reproductive functions in female Sprague-Dawley rats. The Journal of Toxicological Sciences 40: 727–738.

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.

Takahashi M, Inoue K, Morikawa T, Matsuo S, Hayashi S, Tamura K, Watanabe G, Taya K, Yoshida M. 2013. Delayed effects of neonatal exposure to 17alpha-ethynylestradiol on the estrous cycle and uterine carcinogenesis in Wistar Hannover GALAS rats. Reproductive Toxicology 40: 16-23.

U.S. Environmental Protection Agency. 2004. 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).

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