This Event 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.

Key Event: 2365

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Anogenital distance (AGD), increased

Short name

The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. More help

Explore in a Third Party Tool

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help

Level of Biological Organization
Tissue

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place. For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable. For individual/population events, the organ context is not applicable. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Organ term
perineum

Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor). Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Process	Object	Action
androgen receptor signaling pathway	Musculature of female perineum	increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE.Clicking on the name of the AOP will bring you to the individual page for that AOP. More help

AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Androgen receptor agonism leading to long anogenital distance (AGD) in female offspring	AdverseOutcome	Johanna Zilliacus (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 KE.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 in relation to this KE. More help

Term	Scientific Term	Evidence	Link
rat	Rattus norvegicus	High	NCBI
human	Homo sapiens	Low	NCBI

Life Stages

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

Life stage	Evidence
Fetal	High

Sex Applicability

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

Term	Evidence
Female	High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

Anogenital distance (AGD)—the span between the anus and the external genitalia—is a sexually dimorphic trait observed in both rodents and humans, where males exhibit an AGD approximately twice as long as that of females (Liu et al., 2014; Salazar-Martinez et al., 2004; Schwartz et al., 2019; Sharpe, 2020; Thankamony et al., 2016; Wise, 2024). This difference arises from the influence of androgens during fetal development, which drive the formation of secondary sexual characteristics. Presence of androgen in the male fetus drives he elongation of perineum, whereas the lack of androgen in female fetuses prevents this masculinization process (Ipulan et al., 2016; Schwartz et al., 2019; Sharpe, 2020; Welsh et al., 2008).

In rats, androgens act within a critical developmental window – around gestational days 15.5 to 18.5 (Welsh et al, 2008; MacLeod et al., 2010) - and AGD has gained recognition as a reliable proxy for assessing the intrauterine hormonal environment. In XY fetuses, insufficient androgen exposure results in a shorter AGD, while in XX fetuses, excessive androgen exposure can lead to an elongated AGD. This pattern has been observed in both human and rodent studies (Schwartz et al., 2019).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

In rodent studies, anogenital distance (AGD) is measured as the span between the genital papilla and the anus using a stereomicroscope equipped with a micrometer eyepiece. To account for body size, the AGD index (AGDi) is calculated by dividing AGD by the cube root of the body weight.

For statistical analysis, it is essential to treat the litter as the experimental unit, especially when multiple pups from the same litter are assessed. In such cases, statistical models are adjusted by including litter as an independent, random, and nested factor. Additionally, AGD measurements are analyzed using body weight as a covariate, in accordance with the recommendations outlined in OECD Guidance Document 151 (2013).

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). More help

Taxonomic Applicability

The biologically plausible taxonomic applicability domain is mammals, as fetal masculinization is regulated by androgens across all mammalian species (Welsh et al., 2014). The empirical taxonomic applicability domain is based on rat, supported by prenatal studies demonstrating that exogenous androgen treatment leads to a measurable increase in female AGD (Armoskus et al., 2014; Guerra et al., 2014; Hotchkiss et al., 2007; Sathishkumar et al., 2011; Welsh et al., 2009; Wilson et al., 2002; Wolf et al., 2002; Wolf et al., 2004). In humans, epidemiological studies show that patients with polycystic ovary syndrome (PCOS) presents with a longer AGD than controls (Pan et al., 2021; Zamani et al., 2023). There is also some conflicting evidence that girls born to women with PCOS have longer AGD. Evidence regarding AGD in daughters of women with PCOS is mixed: Barrett et al. (2018) reported an association, whereas Glintborg et al. (2019) did not. Additionally, one study found that fetuses of women with PCOS exhibited longer AGD (Perlman et al., 2020).

Sex applicability

This KE focuses on effects on AGD in females. A long AGD in female offspring is a marker of excess (ectopic) androgen action during fetal life (Schwartz et al. 2019). A longer AGD is thus a sign of virilization of the female fetus.

Life Stage Applicability

Growth of AGD in rats is regulated by androgens during the masculinization programming window, which occurs during fetal development between GD15-18 (Welsh et al., 2008; Mc Leod et al, 2010).

Regulatory Significance of the Adverse Outcome

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

Measuring the AGD is mandatory in OECD test guidelines used to test for developmental and reproductive toxicity of chemicals. Guidelines include ‘TG 443 extended one-generation study’ (OECD, 2025a), ‘TG 421/422 reproductive toxicity screening studies’ (OECD, 2025b) and ‘TG 414 developmental toxicity study’ (OECD, 2018). However, there is a huge challenge in interpreting a longer AGD in females.

References

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

Armoskus, C., Mota, T., Moreira, D., & Tsai, H.-W. (2014). Effects of Prenatal Testosterone Exposure on Sexually Dimorphic Gene Expression in the Neonatal Mouse Cortex and Hippocampus. Journal of Steroids & Hormonal Science,5(3), 1000139.

Barrett, E. S., Hoeger, K. M., Sathyanarayana, S., Abbott, D. H., Redmon, J. B., Nguyen, R. H. N., & Swan, S. H. (2018). Anogenital distance in newborn daughters of women with polycystic ovary syndrome indicates fetal testosterone exposure. Journal of Developmental Origins of Health and Disease, 9(3), 307–314. https://doi.org/10.1017/S2040174417001118

Glintborg, D., Jensen, R. C., Schmedes, A. V., Brandslund, I., Kyhl, H. B., Jensen, T. K., & Andersen, M. S. (2019). Anogenital distance in children born of mothers with polycystic ovary syndrome: The Odense Child Cohort. Human Reproduction, 34(10), 2061–2070. https://doi.org/10.1093/humrep/dez122

Guerra, M. T., Silva, R. F., Luchiari, H. R., Sanabria, M., & Kempinas, W. D. G. (2014). Perinatal androgenic exposure and reproductive health effects female rat offspring. Journal of Toxicology and Environmental Health. Part A, 77(7), 375–389. https://doi.org/10.1080/15287394.2013.874881

Hotchkiss, A. K., Furr, J., Makynen, E. A., Ankley, G. T., & Gray, L. E. J. (2007). In utero exposure to the environmental androgen trenbolone masculinizes female Sprague-Dawley rats. Toxicology Letters, 174(1–3), 31–41. https://doi.org/10.1016/j.toxlet.2007.08.008

Ipulan, L. A., Raga, D., Suzuki, K., Murashima, A., Matsumaru, D., Cunha, G., & Yamada, G. (2016). Investigation of sexual dimorphisms through mouse models and hormone/hormone-disruptor treatments. Differentiation; Research in Biological Diversity, 91(4–5), 78–89. https://doi.org/10.1016/j.diff.2015.11.001

Liu, C., Xu, X., & Huo, X. (2014). Anogenital distance and its application in environmental health research. Environmental Science and Pollution Research International, 21(8), 5457–5464. https://doi.org/10.1007/s11356-014-2570-z

MacLeod, D. J., Sharpe, R. M., Welsh, M., Fisken, M., Scott, H. M., Hutchison, G. R., Drake, A. J., & Van Den Driesche, S. (2010). Androgen action in the masculinization programming window and development of male reproductive organs.International Journal of Andrology, 33(2), 279–287. https://doi.org/10.1111/j.1365-2605.2009.01005.x

OECD (2013). Guidance Document Supporting OECD Test Guideline 443 on the Extended One-generation Reproductive Toxicity Test, Series on Testing and Assessment No. 151, https://www.oecd.org/en/publications/guidance-document-on-standardised-test-guidelines-for-evaluating-chemicals-for-endocrine-disruption-2nd-edition_9789264304741-en.html

OECD (2018), Test No. 414: Prenatal Developmental Toxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264070820-en.

OECD (2025a), Test No. 443: Extended One-Generation Reproductive Toxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264185371-en.

OECD (2025b), Test No. 421: Reproduction/Developmental Toxicity Screening Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264380-en.

Pan, Z., Zhu, F., & Zhou, K. (2021). A Systematic Review of Anogenital Distance and Gynecological Disorders: Endometriosis and Polycystic Ovary Syndrome. Frontiers in Endocrinology, 12, 696879. https://doi.org/10.3389/fendo.2021.696879

Perlman, S., Toledano, Y., Kivilevitch, Z., Halevy, N., Rubin, E., & Gilboa, Y. (2020). Foetal Sonographic Anogenital Distance Is Longer in Polycystic Ovary Syndrome Mothers. Journal of Clinical Medicine, 9(9), 2863. https://doi.org/10.3390/jcm9092863

Salazar-Martinez, E., Romano-Riquer, P., Yanez-Marquez, E., Longnecker, M. P., & Hernandez-Avila, M. (2004). Anogenital distance in human male and female newborns: A descriptive, cross-sectional study. Environmental Health, 3(1), 8. https://doi.org/10.1186/1476-069X-3-8

Sathishkumar, K., Elkins, R., Chinnathambi, V., Gao, H., Hankins, G. D. V., & Yallampalli, C. (2011). Prenatal testosterone-induced fetal growth restriction is associated with down-regulation of rat placental amino acid transport. Reproductive Biology and Endocrinology : RB&E, 9, 110. https://doi.org/10.1186/1477-7827-9-110

Schwartz, C. L., Christiansen, S., Vinggaard, A. M., Axelstad, M., Hass, U., & Svingen, T. (2019). Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. Archives of Toxicology, 93(2), 253–272. https://doi.org/10.1007/s00204-018-2350-5

Sharpe, R. M. (2020). Androgens and the masculinization programming window: Human-rodent differences. Biochemical Society Transactions, 48(4), 1725–1735. https://doi.org/10.1042/BST20200200

Thankamony, A., Pasterski, V., Ong, K. K., Acerini, C. L., & Hughes, I. A. (2016). Anogenital distance as a marker of androgen exposure in humans. Andrology, 4(4), 616–625. https://doi.org/10.1111/andr.12156

Welsh, M., Saunders, P. T. K., Fisken, M., Scott, H. M., Hutchison, G. R., Smith, L. B., & Sharpe, R. M. (2008). Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. Journal of Clinical Investigation, 118(4), 1479–1490. https://doi.org/10.1172/JCI34241

Welsh, M., Sharpe, R., Walker, M., Smith, L., & Saunders, P. (2009). New Insights into the Role of Androgens in Wolffian Duct Stabilization in Male and Female Rodents. Endocrinology, 150(5), 2472–2480. (WOS:000265407500056). https://doi.org/10.1210/en.2008-0529

Welsh, M., Suzuki, H., & Yamada, G. (2014). The Masculinization Programming Window. In O. Hiort & S. F. Ahmed (Eds), Endocrine Development (Vol. 27, pp. 17–27). S. Karger AG. https://doi.org/10.1159/000363609

Wilson, V. S., Lambright, C., Ostby, J., & Gray, L. E. J. (2002). In vitro and in vivo effects of 17beta-trenbolone: A feedlot effluent contaminant. Toxicological Sciences : An Official Journal of the Society of Toxicology, 70(2), 202–211. https://doi.org/10.1093/toxsci/70.2.202

Wise, L. D. (2024). Rodent anogenital distance recommendations. Birth Defects Research, 116(6), e2347. https://doi.org/10.1002/bdr2.2347

Wolf, C. J., Hotchkiss, A., Ostby, J. S., LeBlanc, G. A., & Gray, L. E. J. (2002). Effects of prenatal testosterone propionate on the sexual development of male and female rats: A dose-response study. Toxicological Sciences : An Official Journal of the Society of Toxicology, 65(1), 71–86. https://doi.org/10.1093/toxsci/65.1.71

Wolf, C. J., LeBlanc, G. A., & Gray, L. E. J. (2004). Interactive effects of vinclozolin and testosterone propionate on pregnancy and sexual differentiation of the male and female SD rat. Toxicological Sciences : An Official Journal of the Society of Toxicology, 78(1), 135–143. https://doi.org/10.1093/toxsci/kfh018

Zamani, P., Hemati, Z., Kelishadi, R., Kolahdozan, S., Dianatinasab, M., & Keikha, M. (2023). Association between anogenital distance as a noninvasive index in the diagnosis and prognosis of reproductive disorder: A systematic review. International Journal of Reproductive Biomedicine, 21(8), 599–618. https://doi.org/10.18502/ijrm.v21i8.14016