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Relationship: 3718

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 Persistent vaginal cornification

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

Persistent vaginal cornification

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 persistent vaginal cornification via increased kisspeptin release	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 Do not cite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

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.

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.

Vaginal cornification occurs in response to increased estradiol during estrus, and is characterized by a keratinized cell layer (Goldman et al. 2007). Persistent vaginal cornification occurs due to a disruption of the estrus cycle resulting in a prolonged estrus period, and a failure to ovulate.

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 persistent vaginal cornification, in support of development of AOP 623.

Authors of KER 3718 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 ‘Persistent vaginal cornification’ 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 resulting persistent vaginal cornification have been studied in laboratory mammals by addition of various forms of estradiol (e.g. 17beta-estradiol; Kimura 1975; Buchannan et al. 1998; Cooke et al. 1998; Laws et al. 2000; Matsui et al. 2001; Matsuda et al. 2004) and toxicants with endocrine disrupting properties (Ruiz et al. 1996). Studies involving dosing laboratory mammals with various forms of estradiol (e.g. 17beta-estradiol) are supportive of the mechanism of increases in exposure to estradiol compounds causing persistent vaginal cornification. Increased estradiol has been shown to lead to vaginal cornification by binding to estrogen receptors in the epithelium and stroma, leading to proliferation of epithelia cells and production of keratin proteins, with neonatal exposure to estrogen compounds one factor leading to persistent vaginal cornification (Buchanan et al. 1998; Cooke et al. 1998; Masui et al. 2001; Matsuda et al. 2004).

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?	Persistent vaginal cornification?	Summary	Citation
Mice (Mus musculus)	~30 days	50 ug 17B-estradiol daily, injected day 15 or day 17.	yes	yes	Mice fetuses injected with estradiol led to increased frequency of persistent vaginal cornification.	Kimura (1975)
Rats (Rattus norvegicus)	9 days	4 mg/0.2 ml oil Mifepristone daily	yes	yes	Female rats injected with Mifepristone had statistically significant increased plasma estradiol leading to persistent vaginal cornification in all injected animals.	Ruiz et al. (1996)
Mice (Mus musculus)	72 hours	100 ng 17B-estradiol daily, three times.	yes	yes	Female mice injected with estradiol led to vaginal cornification, with tissues with both epithelial (E) and stroma (S) ERα having highest cornification.	Buchanan et al. (1998)
Mice (Mus musculus)	72 hours	100 ng 17B-estradiol daily, three times.	yes	yes	Only female mice injected with estradiol with both epithelial (E) and stroma (S) ERα led to vaginal cornification.	Cooke et al. (1998)
Rats (Rattus norvegicus)	11 days	0.005 mg/kg/d 17B-estradiol, 0.001, 0.01, 0.1 mg/kg/d ethynyl estradiol	yes	yes	All female rats injected with 17B-estradiol or 0.01, 0.1 mg/kg/d ethynyl estradiol led to persistent vaginal cornification	Laws et al. (2000)
Mice (Mus musculus)	55 days	20 ug/g 17B-estradiol daily for 5 days	yes	yes	All female mice injected with estradiol led to persistent vaginal cornification.	Masui et al. (2001)
Mice (Mus musculus)	45 days	0.01, 0.1, 1, 10, 100 ug 17B-estradiol daily for 5 days	yes	yes	All female mice injected with 10, 100 ug estradiol led to persistent vaginal cornification.	Matsuda et al. (2004)

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: Vaginal cornification 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

Buchanan DL, Kurita T, Taylor JA, Lubahn DB, Cunha GR, Cooke PS. 1998. Role of stromal and epithelial estrogen receptors in vaginal epithelial proliferation, stratification, and cornification. Endocrinology 139(10): 4345-4352.

Cooke PS, Buchanan DL, Lubahn DB, Cunha GR. 1998. Mechanism of Estrogen Action: Lessons from the Estrogen Receptor-α Knockout Mouse. Biology of Reproduction 59(3): 470–475.

Goldman JM, Murr AS and Cooper RL. 2007. The rodent estrous cycle: characterization of vaginal cytology and its utility in toxicological studies. Birth Defects Research (Part B) 80: 84-97.

Kimura T. 1975. Persistent vaginal cornification in mice treated with estrogen prenatally. Endocrinologia Japonica 22(6): 497-502.

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.

Masui F, Matsuda M, Akazome Y, Imaoka T, Mori T. 2001. Prevention of neonatal estrogen imprinting by vitamin A as indicated by estrogen receptor expression in the mouse vagina. Cell Tissue Research 306(3): 441-447.

Matsuda M, Masui F, Mori T. Neonatal estrogenization leads to increased expression of cellular retinol binding protein 2 in the mouse reproductive tract. 2004. Cell Tissue Research 316(1): 131-139.

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

Ruiz A, Aguilar R, Tebar AM, Gaytan F, Sanchez-Criado JE. 1996. RU486-treated rats show endocrine and morphological responses to therapies analogous to responses of women with polycystic ovary syndrome treated with similar therapies. Biology of Reproduction 55(6): 1284-1291.

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. 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 February 2026. A full list of updates can be found in the Change Log on the View History page.