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Relationship: 2828

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

Decrease, AR activation leads to Hypospadias

Upstream event

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

Decrease, AR activation

Downstream event

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

Hypospadias

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	Point of Contact	Author Status
Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring	non-adjacent	High	Terje Svingen (send email)	Under development: Not open for comment. Do not cite
Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring	non-adjacent	High	Terje Svingen (send email)	Under development: Not open for comment. Do not cite
5α-reductase inhibition leading to hypospadias in male (mammalian) offspring	non-adjacent	High	Terje Svingen (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 Do not cite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
human	Homo sapiens	High	NCBI
rat	Rattus norvegicus	High	NCBI
mouse	Mus musculus	Moderate	NCBI

Sex Applicability

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

Sex	Evidence
Male	High

Life Stage Applicability

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

Term	Evidence
Foetal	High

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

This non-adjacent KER describes a fetal decrease in androgen receptor (AR) activation in the genital tubercle causing hypospadias in male offspring, postnatally. During fetal development, androgens induce differentiation of the bipotential genital tubercle to a penis, including closure of the urethra. Androgens signal through AR and reduced fetal AR activation can therefore disrupt penis differentiation and lead to the genital malformation hypospadias. Reduced AR activation may happen both through reduced ligand availability (testosterone or dihydrotestosterone (DHT)) and by direct antagonism of AR (Amato et al., 2022; Mattiske & Pask, 2021).

The upstream KE ‘decrease, androgen receptor activation’ (KE 1614) refers to the in vivo event of overall reduction in AR activation. In this case, it therefore refers to a reduction in AR activation in the genital tubercle. Currently, decreased AR activation in mammals is only directly measured in vitro and not in vivo. Instead, indirect assessment of this KE may come from assays measuring AR antagonism, 5α-reductase activity (the enzyme converting testosterone to DHT), or decreased androgen levels (Draskau et al., 2024).

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

A systematic approach (fig. 1, 2d041pib2_KEr_2828_Figure_1.png (1377×385)) was used to collect evidence based on the methodology described in (Holmer et al., 2024).

Search strings were synthesized for PubMed and Web of Science Core Collection based on the review question ‘Does decreased androgen receptor activity during fetal development lead to hypospadias in male mammals?’

Search string in PubMed:

("androgen receptor*" OR "testosterone receptor*" OR "receptors, androgen"[MeSH Terms] OR "androgen*" OR "testosterone*" OR "dihydrotestosterone*" OR "androgens"[MeSH Terms] OR "androgen antagonists"[MeSH Terms]) AND ("genital malformation*" OR "hypospadias"[MeSH terms] OR “hypospadia*”).

Search string in Web of Science Core Collection:

TS=(("androgen receptor*" OR "testosterone receptor*" OR "androgen*" OR "testosterone*" OR "dihydrotestosterone*") AND ("hypospadia*" OR "genital malformation*")).

Title & abstract screening:

Retrieved articles were screened in the online tool RAYYAN https://www.rayyan.ai/. After removal of duplicates, the titles and abstracts of the remaining 1,318 articles were screened according to pre-defined exclusion criteria:

Not English language
Full text not available
Not primary literature
No information about hypospadias in males
Hypospadias was not coupled to reduced AR activity (measured in vitro, by testosterone levels, mutations, known or suspected anti-androgenic exposure)

During this screening, included papers were split into animal studies and human case studies, respectively.

Full text review, data extraction and reliability evaluation of animal studies:

For animal studies, the full text papers were reviewed using the same exclusion criteria as in the title & abstract screening, and data were extracted from the included papers into an Excel template. In parallel, methodological reliability was assessed using the online tool Science in Risk Assessment and Policy (SciRAP; http://www.scirap.org, see appendix 1, 431r6dhi30_KER_2828__appendix_1.pdf). Based on the SciRAP evaluations, the animal studies were assigned a reliability category using the principles outlined in table 1. Studies were divided into different datasets, if exposure scenarios led to assignment to different reliability categories. Only studies in reliability categories 1 (reliable without restriction) and 2 (reliable with restriction) were used for the assessment of overall confidence in the data. The animal studies were grouped according to the model substances used. Model substances were selected to include different upstream effects that are known to reduce AR activity: Flutamide, dibutyl phthalate (DBP), vinclozolin, di(2-ethylhexyl) phthalate (DEHP), procymidone, and finasteride.

For each chemical, the overall confidence in the collected data was assessed according to the principles outlined in table 2.

Table 1 Principles for translation SciRAP evaluations into reliability categories.

Reliability Category	Principles for Categorization
1.Reliable without restriction	SciRAP methodological quality Score > 80 and all key criteria^a are “Fulfilled” and there are no deficiencies in the non-key criteria that might affect study reliability.
2. Reliable with restriction	SciRAP methodological quality Score > 65 and one or several of the key criteria are “Partially Fulfilled” or there are minor deficiencies in the non-key criteria that might affect study reliability.
3. Not reliable	SciRAP methodological quality Score < 65 or one or several of the key criteria are “Not Fulfilled” or there are major deficiencies in the non-key criteria that affect study reliability.
4. Not assignable	Two or more of the key criteria are “Not Determined”

^aKey criteria were criteria judged as specifically critical for reliability of the data for this KER and were determined a priori. The key criteria for this data collection are outlined in appendix 1, 431r6dhi30_KER_2828__appendix_1.pdf.

Table 2 Principles for evaluation of overall confidence in data for each selected substance.

Level of confidence	Principles for Categorization^a
Strong	Effects were observed in one or more studies judged as reliable without restriction or reliable with restriction; there are no conflicting results from studies judged as reliable with or without restriction. OR Effects were observed in one or more studies judged as reliable without restriction or reliable with restriction but conflicting results, i.e. no or opposite effects were observed in other studies judged as reliable with or without restriction. However, conflicts of results can be explained by differences in study design, for example different exposure periods, doses or animal species or cell models.
Moderate	Effects were observed in one or more studies judged as reliable without restriction or reliable with restriction but conflicting results, i.e., no or opposite effects were observed in other studies judged as reliable with or without restriction. Conflicts of results cannot be explained by differences in study design, for example different exposure periods, doses or animal species or cell models. Effects were observed in at least half of the studies.
Weak	Effects were observed in one or more studies judged as reliable without restriction or reliable with restriction but conflicting results, i.e., no or opposite effects were observed in other studies judged as reliable with or without restriction. Conflicts of results cannot be explained by differences in study design, for example different exposure periods, doses or animal species or cell models. Effects were observed in fewer than half of the studies. OR Effects were only observed in one or more studies judged as not reliable or not assignable.
No effect	No effects were observed in any of the studies reviewed.

^a Conflicting results from studies judged as not reliable do not impact categorization.

Full text review and data extraction of human studies

For studies in humans, regarded as supporting evidence, the full text papers were reviewed using the same exclusion criteria as in the title & abstract screening, and data were extracted from the included papers into an Excel template. For these studies, the occurrence of hypospadias and the underlying cause were the main data points extracted. The papers were then grouped according to the underlying condition of the reported patients. Four main groups of upstream effects were reported, which were then split into more specific sub-groups:

Effects on AR: This included mutations in AR, extended CAG repeat length in AR, which is associated with reduced AR activity (Chamberlain et al., 1994), and studies in which low AR activity was measured in vitro in genital skin fibroblasts from hypospadias patients.

Effects on 5α-reductase activity: This included mutations in SRD5A2, high testosterone/DHT ratio, and studies in which low 5α-reductase activity was measured in vitro in genital skin fibroblasts from hypospadias patients.

Effects on upstream steroidogenesis: This included mutations in steroidogenesis enzymes (HSD17B3, HSD3B2, or CYP17A1) and patients diagnosed with deficiency in these enzymes based on their hormone and metabolite profile.

Other upstream effects: These were studies, in which low testosterone (basal or hCG-stimulated) was measured in patients, either idiopathic or due to gonadal dysfunction or rare mutations.

The final list of studies was based on an assessment of whether there was a clear description of conditions with decreased AR activity (as described above) and associated hypospadias. Moreover, epidemiologic and case-control studies were included as supporting evidence, and these were included, even if they did not show a causal relationship between reduced AR activity and hypospadias.

Exploration of taxonomic domain of applicability

A separate literature search was performed in “Web of Science, All Databases” to explore the taxonomic domain of applicability of the KER in relation to wildlife species and non-mammalian vertebrates. These studies were not systematically evaluated for reliability but served as supporting evidence for the taxonomic applicability domain of the KERs.

Search string in Web of Science all databases:

hypospadia*and (wildlife OR “animal model” OR domestic OR evolution*).

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

The biological plausibility for this KER is judged as high. This is largely based on canonical knowledge on normal reproductive development.

The penis originates from a sexually bipotential structure, the genital tubercle, which may differentiate to either a penis or a clitoris, depending on internal cues during fetal development. In males, the fetal testes produce large amounts of testosterone, which can subsequently be converted to the more potent androgen DHT by 5α-reductase in peripheral tissues. Testosterone and DHT both signal through AR in target tissues to initiate masculinization (Amato et al., 2022; Murashima et al., 2015). The critical developmental window for androgen programming of masculinization has been identified in rats as GD16-20, and is proposed to be gestational weeks 8-14 in humans (Sharpe, 2020; Welsh et al., 2008). As part of the masculinization process orchestrated by androgens, the genital tubercle differentiates to a penis, which at this point expresses AR in both humans and rodents (Amato & Yao, 2021; Baskin et al., 2020). This includes androgen-mediated elongation of the tubercle, formation of the prepuce, and tubular internalization of urethra, which is closed at the distal tip of the glans penis (Amato et al., 2022). Failure of full closure of the urethra can result in hypospadias, in which the urethra terminates at the ventral side of the penis instead of at the tip (Baskin & Ebbers, 2006; Cohn, 2011).

The dependency of androgens for penile development has been demonstrated in mice with conditional or full knockout of Ar, which results in partly or full sex-reversal of males, including a female-like urethral opening (Willingham et al., 2006; Yucel et al., 2004; Zheng et al., 2015). Similarly, female rats and mice exposed in utero to testosterone present with varying degrees of intersexuality, including, in some cases, a penis (Greene & Ivy, 1937; Zheng 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

The empirical evidence for this KER is generally judged high. This includes evidence from in vivo animal studies and evidence from studies in humans. The upstream KE ‘Decreased AR activity’ refers to an in vivo effect, for which no methods for measurement of this in vivo in mammals currently exist. The effects on the upstream KE were therefore indirectly informed as described in each section.

Animal studies

Effects on the upstream KE were indirectly informed by including animal studies with stressors that are known to reduce AR activity through antagonizing the AR, lowering testosterone production, or inhibiting 5α-reductase. Six stressors, with established anti-androgenic effects, were included (more detailed evaluation of these chemicals can be found in KER-2820 (Holmer et al., 2024)). Table 3 summarizes the empirical evidence and confidence level for each chemical. Details on included evidence is presented in Table 1 in Appendix 2, 9prbqyba2x_Appendix_2_KER_2828.pdf. In summary, all six substances were shown to cause hypospadias in male offspring, and the confidence level for all substances was judged as strong, as conflicting results could be explained (see the section ‘Uncertainties and inconsistencies’). Thus, antagonism or AR, inhibition of 5α-reductase, or reduction in testosterone synthesis, all lead to hypospadias.

Table 3 Summary of empirical evidence for the KER – animal studies. See Table 1 in Appendix 2 ( 9prbqyba2x_Appendix_2_KER_2828.pdf) for details.

Chemical	Upstream effect	Downstream effect	Overall confidence
Flutamide	Androgen receptor antagonist (Simard et al., 1986).	In utero exposure causes hypospadias in rat and mouse	Strong
Dibutyl phthalate (DBP)	Has been shown to reduce fetal intratesticular testosterone and serum testosterone in vivo, but exact mechanism is unknown (Foster, 2006).	In utero exposure causes hypospadias in rat	Strong
Vinclozolin	AR antagonist (Kelce et al., 1994, 1997)	In utero exposure causes hypospadias in rat and mouse	Strong
Di(2-ethylhexyl) phthalate (DEHP)	Has been shown to reduce fetal intratesticular testosterone and serum testosterone in vivo, but exact mechanism is unknown (Parks et al., 2000).	In utero exposure causes hypospadias in rat	Strong
Procymidone	AR antagonist (Ostby et al., 1999).	In utero exposure causes hypospadias in rat	Strong
Finasteride	5α-reductase inhibitor, causing a reduction in DHT (Rittmaster & Wood, 1994).	In utero exposure causes hypospadias in rat	Strong

Supporting human evidence

Effects on the upstream KE were indirectly informed by including studies in humans with a condition (genetic or other) that would reduce or disrupt either 1) function of AR, 2) conversion of testosterone to DHT by disrupting 5α-reductase activity, or 3) production of androgen hormones. Studies measuring low testosterone levels with no underlying cause were also included (see evidence collection strategy). Table 4 lists the studies, in which these conditions were linked to hypospadias in males.

Table 4 Supporting evidence for the KER – human studies. The table lists human studies reporting hypospadias in association with an upstream defect in AR activity, grouped according to the precise effect, and how it was diagnosed (mutation, in vitro activity, or blood hormone and metabolite profile). SRD5A2: 5α-reductase 2; HSD17B3: 17β-hydroxysteroid dehydrogenase 3; HSD3B2: 3β-hydroxysteroid dehydrogenase 2; CYP17A1: 17α-hydroxylase. See table 2 in Appendix 2 (9prbqyba2x_Appendix_2_KER_2828.pdf) for all included references.

Effect on upstream KE	Supporting studies
Effects on Androgen receptor
AR mutations	27 studies
Extended CAG repeat length in AR	4 studies
Reduced AR activity (e.g. low receptor binding) in in vitro genital skin fibroblasts	11 studies
Effects on 5α-reductase activity
SRD5A2 mutations	30 studies
SRD5A2 deficiency, diagnosed by T/DHT-ratio and/or reduced in vitro 5α-reductase activity in genital skin fibroblasts	8 studies
Effects on upstream steroidogenesis enzymes
HSD17B3 mutations	6 studies
HSD3B2 mutations	5 studies
CYP17A1 mutation	1 study
HSD17B3 deficiency, diagnosed by hormone and metabolite profile	2 studies
HSD3B2 deficiency, diagnosed by hormone and metabolite profile	4 studies
CYP17A1 deficiency, diagnosed by hormone and metabolite profile	5 studies
Other upstream effects on low testosterone
Low testosterone due to gonadal dysgenesis or hypogonadism	7 studies
Low basal testosterone or low testosterone response to hCG stimulation. Idiopathic or rare mutations.	7 studies

Six case-control studies were extracted, all of which found a correlation between lower testosterone levels (basal or hCG-stimulated) and hypospadias (Austin et al., 2002; Okuyama et al., 1981; Raboch et al., 1976; Ratan et al., 2012; Svensson et al., 1979; Yadav et al., 2011). In two of these studies, the correlation was age-dependent (Austin et al., 2002; Raboch et al., 1976)

One epidemiologic study was extracted, which investigated the association between phthalate exposure and hypospadias risk. Western Australian women exposed through their occupation to phthalates were more likely to have sons with hypospadias (Nassar et al., 2010). It should be noted that there are reported species differences in the effects of phthalates (including DEHP and DBP) on fetal testosterone production between humans, mice, and rats, and the direct translatability of the in vivo evidence is uncertain (Sharpe, 2020).

Dose concordance

Direct information about dose concordance is not available because AR activity currently cannot be measured in vivo.

Indirect information on dose concordance can be obtained from empirical evidence. In utero exposure of rats to DBP caused a dose-dependent decrease in serum testosterone levels at PND70 with LOAEL 250 mg/kg bw/day. Hypospadias was observed at this stage with LOAEL 500 mg/kg bw/day (Jiang et al., 2007). It should be noted that fetal testosterone levels were not measured.

Temporal concordance

Direct information about temporal concordance is not available because AR activity currently cannot be measured in vivo.

Indirect information on temporal concordance can be obtained from empirical evidence. In two studies, in which rats were exposed in utero to 750 mg/kg bw/day DBP, intratesticular testosterone levels were reduced in fetal testes, while hypospadias was identified in adult males. Plasma levels of testosterone were also measured in adults, and testosterone levels in exposed males were not significantly different from control males (van den Driesche et al., 2017, 2020). This has also been shown in a study with 500 mg/kg bw/day DBP (Drake et al., 2009). These studies indicate temporal concordance. Another study with DBP-induced hypospadias in rats saw a dose-dependent reduction in serum testosterone levels at PND70 after in utero exposure to as low as 250 mg/kg bw/day from GD14-18 (Jiang et al., 2007), though fetal testosterone levels were not measured in this study.

Incidence Concordance

Direct information about dose concordance is not available because AR activity currently cannot be measured in vivo.

Indirect information on incidence concordance can be obtained from empirical evidence. In the dose-response study with DBP, the incidence of hypospadias was 6.8% for 500 mg/kg bw/day DBP and 41.3% for 750 mg/kg bw/day. When separating hypospadias males from exposed males without hypospadias, plasma testosterone levels were decreased in both groups, indicating that DBP reduced testosterone levels at higher incidence than hypospadias (Jiang et al., 2007). The same was seen in another study with DBP, in which serum testosterone levels at PND7 were reduced in both hypospadiac and non-malformed males exposed to 750 mg/kg bw/day DBP from GD14-18 (Jiang et al., 2016).

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

The in vivo studies do not directly inform about the upstream KE, ‘decreased AR activity’. The direct concordance between the KEs can therefore not be determined from the evidence.

For flutamide, two studies reported 100% hypospadias frequencies at doses of 6.25 and 10 mg/kg bw/day (Goto et al., 2004; McIntyre et al., 2001), while another study found a frequency of 56.9% when giving 20 mg/kg bw/day (Kita et al., 2016). This might be explained by a longer exposure window in the first two studies and uncertainties in assessment of hypospadias.

For DBP, there were discrepancies in whether 250 mg/kg bw/day was LOAEL (Mylchreest et al., 1998, 1999) or NOAEL (Jiang et al., 2007) for DBP. This conflict was explained by differences in exposure windows, supported by the observation that the frequency of hypospadias at 250 mg/kg bw/day was reported as very low (Mylchreest et al., 1998, 1999).

One study with vinclozolin (Ostby J et al., 1999) and one with procymidone (Hass et al., 2012) did not find hypospadias after in utero exposure. In both cases, this was likely due to too low doses tested.

In most of the human studies of steroidogenesis deficiency, serum or plasma levels of testosterone were reduced at baseline and/or upon hCG stimulation (Al-Sinani et al., 2015; Ammini et al., 1997; Cara et al., 1985; Chen, Huang, et al., 2021; Dean et al., 1984; Galli-Tsinopoulou et al., 2018; Imperato-McGinley et al., 1979; Kaufman et al., 1983; Mendonca et al., 1987, 2000; Neocleous et al., 2012; New, 1970; Pang et al., 1983; Perrone et al., 1985; Rabbani et al., 2012; Sherbet et al., 2003), but in a few studies, testosterone levels were normal (Donadille et al., 2018; Kon et al., 2015; Luna et al., 2021). In these cases, the effect of these deficiencies on tissue AR activity is uncertain.

For AR CAG repeat length, a case-control study did not find an association with hypospadias (Radpour R et al., 2007), but this could be because the hypospadias cases included had other etiologies.

Lastly, as there are currently no universal guidelines for identification and scoring of hypospadias in rodents, there are large variations in methods of assessment, and minor cases of hypospadias may be overlooked in some studies and included in others. This poses an uncertainty in the frequency reports in the scientific evidence.

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

Modulating Factors	MF details	Effects on the KER	References
AR CAG repeat length	Extended CAG repeat length in AR is associated with reduced AR activity	Higher risk of hypospadias development	(Chamberlain et al., 1994)

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

The quantitative understanding of the relationship is low. As there are currently no direct measurement methods of the upstream KE (reduced AR activity) in mammals, quantification of the relationship is difficult to assess.

Response-response Relationship

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

A model for phthalates has been developed, aiming to predict the frequency of hypospadias in male offspring based on reductions in ex vivo testosterone production, an indirect indication of AR activity. In this model, hypospadias was induced from around a 60% reduction in testosterone levels. The model does not consider hypospadias severity and is only for phthalate chemicals (Earl Gray et al., 2024).

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

The time-scale of this KER depends on the species but is likely days to weeks.

AR activation happens within minutes, from ligand binding to nuclear translocation and promotor activation (Nightingale et al., 2003; Schaufele et al., 2005), while transcriptional and translational effects are observed minutes to hours later (Kang et al., 2002). AR programming of the genital tubercle occurs during fetal development in the Masculinization Programming Window (Sharpe, 2020). The time-scale for morphological effects in the tissue then depends on the species. In humans, penis development is completed prior to birth and hypospadias can be observed at birth. In rodents, penis development is not fully completed until weeks after birth, but hypospadias can often be observed earlier than this (table 3).

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

There are no known feedforward/feedback loops influencing this KER.

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

Taxonomic applicability

In mammals, androgens are one of the primary drivers of penis differentiation. Hypospadias has been observed in several mammals, but most frequently reported in laboratory rodents and in humans (Chang et al., 2020; S. Wang & Zheng, 2025). In vivo studies in rats and mice show that in utero exposure to anti-androgenic chemicals can cause hypospadias in male offspring (see table 3). Many human case studies report boys born with hypospadias and associated deficiency in steroid hormone synthesis, 5α-reductase activity, or androgen receptor (AR) activity (see table 4).

The biologically plausible domain of applicability may extend beyond the empirical domain because androgen-controlled development of male external genitalia is evolutionary conserved in most mammals and, to some extent, also in other vertebrate classes (Gredler et al., 2014). Hypospadias can in principle occur in all animals that form a genital tubercle and have been observed in many domestic animal species including dog (Sonne et al., 2008; Switonski et al., 2018), cat (Nowacka-Woszuk et al., 2014), cattle (Murakami, 2008), sheep (Smith et al., 2012), and horse (De Lorenzi et al., 2010) as well as in wildlife species such as polar bear (Stamper et al., 1999), giraffe (Meuffels et al., 2020), and Tamar Wallaby (Leihy et al., 2011). The observed hypospadias in these animals is not, per se, linked to anti-androgenic exposure, which has only been sparsely investigated in other species than mice, rats, and humans. One study in monkeys did show hypospadias upon oral exposure to finasteride (Prahalada et al., 1997), and bicalutamide exposure induced hypospadias in guinea pigs (S. Wang et al., 2018). A study in rabbits exposed to procymidone did not find hypospadias in males (Inawaka et al., 2010). Another study in hyenas did also not find hypospadias in males after exposure to the anti-androgen finasteride (Drea et al., 1998), but it should be noted that the hyenas have a remarkable sexual development where penile growth occur in both females and males before androgen synthesis is initiated (Cunha et al., 2014) (the studies in hyena and rabbit were identified in our evidence collection but were judged as ‘unreliable’ and therefore not included as empirical evidence).

Sex applicability

The androgen receptor is expressed in the fetal genital tubercle of both females and males (Amato & Yao, 2021; Baskin et al., 2020), but hypospadias is primarily a term used for a malformation of the penis (Baskin & Ebbers, 2006), limiting the applicability of this KER to males.

Life stage applicability

Differentiation of the penis occurs during fetal life in the masculinization programming window (MPW) (GD 16-20 in rats, around gestational weeks 8-14 in humans), when androgen production is high (Welsh et al., 2008; C. Wolf et al., 2000a). In rats, exposure to anti-androgenic chemicals outside of, or in the late part of the MPW does not cause hypospadias or only to a low degree (Clark et al., 1993; van den Driesche et al., 2017; C. Wolf et al., 2000a), while exposure in the earlier (or full) MPW causes a higher frequency of hypospadias (depending on dose and chemical) (table 3). In humans, hypospadias can be diagnosed at birth (X. Yu et al., 2019), while in rodents, some parts of penis development occur postnatally (Schlomer et al., 2013; Sinclair et al., 2017). In these species, hypospadias may be observed at birth but is optimally diagnosed and severity classified weeks later. Given that disruptions to androgen programming takes place in fetal life, even though the AO is best detected postnatally, the life stage applicability is defined as fetal life.

References

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

Al-Sinani, A., Mula-Abed, W., Al-Kindi, M., Al-Kusaibi, G., Al-Azkawi, H., & Nahavandi, N. (2015). A Novel Mutation Causing 17-β-Hydroxysteroid Dehydrogenase Type 3 Deficiency in an Omani Child: First Case Report and Review of Literature. Oman Medical Journal, 30(2), 129–134. https://doi.org/10.5001/omj.2015.27

Amato, C. M., & Yao, H. H.-C. (2021). Developmental and sexual dimorphic atlas of the prenatal mouse external genitalia at the single-cell level. Proceedings of the National Academy of Sciences of the United States of America, 118(25). https://doi.org/10.1073/pnas.2103856118

Amato, C. M., Yao, H. H.-C., & Zhao, F. (2022). One Tool for Many Jobs: Divergent and Conserved Actions of Androgen Signaling in Male Internal Reproductive Tract and External Genitalia. Frontiers in Endocrinology, 13, 910964. https://doi.org/10.3389/fendo.2022.910964

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