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Relationship: 3349

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, circulating testosterone levels leads to AGD, decreased

Upstream event

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

Decrease, circulating testosterone levels

Downstream event

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

AGD, decreased

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
Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring	non-adjacent	High	Moderate	Terje Svingen (send email)	Under development: Not open for comment. Do not cite	Under Review

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		NCBI
rat	Rattus norvegicus	High	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 circulating testosterone (often measured in serum or plasma) leading to short anogenital distance (AGD) in male offspring.

In male mammals, testosterone is one of the primary hormonal drivers of male reproductive differentiation. Produced by the fetal testes, testosterone is transported through blood to the peripheral reproductive tissues to bind the androgen receptor (AR) or be converted to the higher potency androgen hormone dihydrotestosterone (Murashima et al., 2015). The androgen hormones signal through AR to program the reproductive tissue to differentiate along the male pathway. This includes elongation of the perineum, which is suggested to involve the perineal muscle complex levator ani bulbocavernous (LABC). LABC expresses AR and increases in size by androgen programming (Schwartz CL et al., 2019). The male programming of the tissue happens during fetal life in the masculinization programming window (GD 16-20 in rats, GW 8-14 in humans), when circulating testosterone levels are high (Sharpe RM, 2020; Welsh M et al., 2014). Thus, a decrease in circulating testosterone levels in this window may limit the AR signaling in the LABC, leading to less elongation of the perineum and a short AGD.

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 was used to collect evidence based on the methodology described in (Holmer et al., 2024). The evidence collection for this KER was done concurrently with the evidence collection for KER 3449 ‘decreased intratesticular testosterone leads to decreased AGD’, for which the same search string was used. See figure 1: 6ieml3mmfa_Figure_1.pdf

Search strategy

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

Search string in PubMed: "testosterone*" AND ("anogenital distance*” OR “AGD”)

Search string in Web of Science Core Collection: "testosterone*" AND ("anogenital distance*” OR "AGD")

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 649 articles were screened according to pre-defined inclusion and exclusion criteria:

Inclusion criteria:

In vivo studies in male mammals where fetal testosterone is reduced and AGD is measured^*
Reviews on AGD
Epidemiologic studies with measurement of testosterone levels and AGD as an outcome
In vitro, ex vivo, and in vivo mechanistic studies on AGD

Exclusion criteria:

Papers not in English
Abstracts and other non-full text publications

^*In cases where this criterion could not be determined by reading the abstract, the full texts were checked in the reference manager Zotero to determine if the testosterone levels were reduced, and when the measurements were made.

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

For the in vivo 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: 2guzo63o2d_KER_3349_Appendix_1.pdf). Based on the SciRAP evaluations, animal studies were assigned a reliability category using the principles outlined in table 1. Studies were divided into different datasets, if multiple different chemicals, different exposure windows, or different time points of measurement of AGD were included.

Moreover, as this KER was made in parallel with several other KER for other male reproductive endpoints (nipple retention and hypospadias), eight studies retrieved in the searches for these KERs which also measured AGD, but were not detected in the search for this KER were also added, data extracted and evaluated for reliability.

The collected data was then filtered to only include data sets measuring circulating testosterone, either in plasma or serum.

Overall confidence in the collected data was assessed according to the principles outlined in table 2. 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.

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.

Table 2 Principles for evaluation of overall confidence in data

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.

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 to be high given the canonical biological knowledge on normal reproductive development.

Sexual differentiation in males, including elongation (masculinization) of the perineum, is initiated and programmed in fetal life. Once the testes have formed, they start producing testosterone through the steroidogenesis pathway and secrete testosterone into circulation. Testosterone is transported in the blood either as free testosterone or bound to albumin or sex-hormone binding globulin. In peripheral tissues, testosterone can be converted to the more potent androgen hormone dihydrotestosterone (DHT) by the enzyme 5α-reductase. Both DHT and testosterone bind and activate the androgen receptor (AR) to program fetal tissues to differentiate along the male pathway, including elongation of the perineum, resulting in a longer AGD in males than in females (~twice the length in rats and humans) (Murashima et al., 2015; Trost & Mulhall, 2016; Welsh M et al., 2014)

Testosterone is produced from around GD15 in fetal rats and GW8 in humans, which is also the onset of when testosterone levels can be measured in circulation. The programming of the reproductive tissues, including masculinization of the perineum happens in the masculinization programming window (GD16-20 in rats, GW8-14 in humans) (Welsh M et al., 2014).

Given the dependency of testosterone for elongation of the perineum, either through direct AR activation or conversion to DHT, it is highly plausible that a decrease in circulating levels of testosterone will lead to a shorter AGD in males

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 from studies in animals for this KER is overall judged as strong.

From the data collection, ten data sets were extracted. The data sets included different stressors causing reduced fetal levels of testosterone, all in rats (table 3 and appendix 2: 99hykht8a4_KER_3349_Appendix_2.pdf). Of these ten data sets, eight showed concurrent short AGD.

Table 3 Empirical evidence for KER 3349 LOAEL: Lowest observed adverse effect level; NOAEL: No observed adverse effect level. See appendix 2 for specifications.

Species	Stressors(s)	Effect on upstream event (circulating testosterone)	Effect on downstream event (AGD)¹	Reference
Rat	2,3,7,8-Tetrachlorodibenzo-p-dioxin	LOAEL 1 µg/kg	LOAEL 1 µg/kg	(Mably TA et al., 1992)
Rat	Dicyclohexyl phthalate	LOAEL 100 mg/kg	LOAEL 20 mg/kg	(Aydoğan Ahbab M & Barlas N, 2015)
Rat	Diethylhexyl phthalate	LOAEL 750 mg/kg	LOAEL 750 mg/kg	(Borch J et al., 2004)
Rat	Diethylhexyl phthalate + Diethylhydroxylamine	LOAEL 750 + 400 mg/kg	LOAEL 750 + 400 mg/kg	(Borch J et al., 2004)
Rat	Di-n-hexyl phthalate	LOAEL 20 mg/kg	Short AGD at 20 and 500 mg/kg, but not 100 mg/kg	(Aydoğan Ahbab M & Barlas N, 2015)
Rat	Perfluorotridecanoic acid	LOAEL 1 mg/kg	LOAEL 10 mg/kg	(Li C et al., 2021)
Rat	Prochloraz	LOAEL 30 mg/kg	No effect NOAEL 30 mg/kg	(Vinggaard AM et al., 2005)
Rat	Prochloraz	LOAEL 50 mg/kg¹	LOAEL 50 mg/kg	(Laier P et al., 2006)
Rat	Zearalenone	LOAEL 10 mg/kg	LOAEL 5 mg/kg	(Pan P et al., 2020)
Rat	Mixture (prochloraz, deltamethrin, methiocarb, simazine, tribenuron)	LOAEL 20 mg/kg	No effect NOAEL 20 mg/kg	(Vinggaard AM et al., 2005)

¹NOAEL and LOAEL were, when available, based on AGDi data. For some datasets, only AGD or AGD/bw were available, see appendix 2 for details on each dataset.

²No statistics available as samples were pooled for measurement of testosterone.

Supporting epidemiological evidence

No studies have shown a direct association between fetal circulating testosterone levels and AGD. A few epidemiologic studies can inform indirectly on the human evidence for this KER, and the current studies on this are conflicting.

As some phthalates are known to reduce testosterone production, an association between phthalate exposure and short AGD could support the KER in humans. A meta-analysis found an association between maternal urinary concentrations of some phthalate metabolites and short AGD (Zarean M et al., 2019). Moreover, in a Taiwan Maternal and Infant Cohort study, maternal urinary concentrations of some phthalate metabolites were also associated with a shorter AGD in male infants, although there was no association between cord blood testosterone levels and AGD, and the metabolites were not associated with lower cord blood testosterone levels, either (Lu et al., 2024). Another longitudinal mother-child cohort study did not find an association between AGD in adult men with maternal serum concentrations of phthalate metabolites during pregnancy (Henriksen LS et al., 2023).

One study related cord blood testosterone levels to AGD in infants boys and did not find associations between the two (Liu C et al., 2016).

In adult men, anogenital distance was significantly associated with serum testosterone levels (Eisenberg ML et al., 2012).

Dose concordance

The in vivo rat toxicity studies for this KER moderately supports dose concordance.

One study with the stressor perfluorotridecanoic acid showed dose concordance with the LOAEL for reduced serum testosterone being 1 mg/kg bw/day, while the LOAEL for short AGD was 10 mg/kg (Li C et al., 2021).

In another study with two doses of prochloraz, the LOAEL was the same (50 mg/kg) for decreased testosterone and short AGD (Laier P et al., 2006).

Finally, in two studies with dicyclohexyl phthalate and zearalenone, respectively, AGD was shortened at lower doses than testosterone was reduced (Aydoğan Ahbab M & Barlas N, 2015; Pan P et al., 2020), and these do therefore not support dose concordance. However, in both cases the testosterone levels tended to be lower in the non-significant doses as well, and the lack of effect could therefore be due to high variation in the testosterone measurements.

Temporal concordance

Overall, the empirical evidence supports temporal concordance between the events.

Mably TA et al., 1992 followed plasma testosterone levels in male rats after in utero exposure to 1 µg/kg 2, 3, 7, 8-Tetrachlrodibenzo-p-dioxin on GD15. From GD17-21, testosterone levels steadily decreased in both control and exposed males, but the overall levels in the exposed males were lower than in control males. After birth, the testosterone levels in exposed male increased to match values in control males. At PND1, 3, and 5, plasma levels were normal in exposed male rats, while their AGD were reduced at all days.

Five of the data sets also measure circulating testosterone prenatally (GD20 or GD21) and AGD postnatally (between PND0 and PND3), and three of these observe short AGD when testosterone is reduced during fetal life (Borch J et al., 2004; Laier P et al., 2006). In one of these studies, where diethylhexyl phthalate alone or diethylhexyl phthalate in combination with diethylhydroxylamine was administered from GD7-PND17, serum testosterone levels were decreased at GD21, but not at PND22 or PD90 (Borch J et al., 2004). Similarly, PND16 serum testosterone was not altered by perinatal (GD7-PND16) prochloraz exposure which reduced serum testosterone at GD21 and AGD at PND1 (Laier P et al., 2006).

Incidence concordance

The data does not inform incidence concordance.

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

Two data sets, both from the same study (Vinggaard AM et al., 2005), showed no effect of decreased circulating testosterone levels on AGD, which may be due to too low doses of the stressors (prochloraz and a mixture). For two studies (Aydoğan Ahbab M & Barlas N, 2015; Pan P et al., 2020), the LOEAL was lower for the downstream event, short AGD, than the upstream event, reduced circulating testosterone. In both cases, lower doses of stressors tended to lower testosterone levels as well, and the inconsistency could therefore be due to high variance in testosterone measurements.

Another uncertainty is the AGD results in the study investigating di-n-hexyl phthalate exposure from GD6-19 in rats (Aydoğan Ahbab M & Barlas N, 2015). Three doses of the phthalate (20, 100, and 500 mg/kg bw/day) all reduced plasma testosterone levels, but only 20 and 500 mg/kg bw/day caused short AGD, when calculating the anogenital distance index (AGDi, AGD/bw^1/3). When analyzing the direct AGD, all doses of di-n-hexyl phthalate decreased AGD. In contrast, when analyzing the relative AGD (AGD/bw), only the highest dose (500 mg/kg bw/day) decreased the AGD. This study thus identified different LOAELs for AGD, depending on if and how body weight was considered, posing an uncertainty on the results.

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

There are no known modulating factors for this KER.

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

Testosterone is secreted from around GW8 in humans (GD16 in rats), marking the beginning of the masculinization programming window and programming of the perineal tissue. Depending on the species, the time scale for observing effects on tissue growth is days or weeks. In humans, sexual dimorphism of the AGD can be measured by GW11-13, reaching the full 2:1 male:female ratio in length at GW17-20 (Thankamony A et al., 2016).

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 feedback/feedforward loops for 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

Male-specific development of the fetal perineum in male mammals is strongly influenced by androgen signaling. It is therefore biologically plausible that this KER is applicable to all mammals (Murashima et al., 2015). The empirical evidence in this KER provides strong support that reduced circulating testosterone levels in fetal life can cause short AGD in rats. The empirical evidence for this KER in humans is sparse and conflicting; however, given the known role of androgens in human male reproductive development, the KER is considered applicable to humans.

Sex applicability

The empirical evidence in this KER supports that reduced circulating testosterone in fetal life can cause reduced AGD in males. Females do have circulating testosterone, but in much lower concentrations than males (Vesper et al., 2015), and it is unlikely that further reduction can cause a short AGD in females (Schwartz CL et al., 2019). Of note is that ‘reduced AGD’ in males is not a reduction per se, but a failure to elongate in response to androgen action.

Life stage applicability

This KER is applicable to fetal life, as this is when the perineum is programmed by androgen hormones in males. The masculinization programming window is around gestational days (GD) 16-20 in rats, and suggested to be gestational weeks (GW) 8-14 in humans (Sharpe RM, 2020; Welsh M et al., 2014). Once programmed in fetal life, the AGD is believed to be relatively stable, but the perineum can in some cases be responsive to postnatal changes in androgen levels (Schwartz CL et al., 2019; Sharpe RM, 2020; Thankamony A et al., 2016). The empirical evidence in this KER supports the fetal life stage applicability.

References

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

Aydoğan Ahbab M & Barlas N. (2015). Influence of in utero di-n-hexyl phthalate and dicyclohexyl phthalate on fetal testicular development in rats. Toxicology Letters, 233(2), 125–137. https://doi.org/10.1016/j.toxlet.2015.01.015

Borch J, Ladefoged O, Hass U, & Vinggaard AM. (2004). Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats. Reproductive Toxicology (Elmsford, N.Y.), 18(1), 53–61. https://doi.org/10.1016/j.reprotox.2003.10.011

Eisenberg ML, Jensen TK, Walters RC, Skakkebaek NE, & Lipshultz LI. (2012). The relationship between anogenital distance and reproductive hormone levels in adult men. The Journal of Urology, 187(2), 594–598. https://doi.org/10.1016/j.juro.2011.10.041

Henriksen LS, Frederiksen H, Jørgensen N, Juul A, Skakkebæk NE, Toppari J, Petersen JH, & Main KM. (2023). Maternal phthalate exposure during pregnancy and testis function of young adult sons. The Science of the Total Environment, 871, 161914. https://doi.org/10.1016/j.scitotenv.2023.161914

Holmer, M. L., Zilliacus, J., Draskau, M. K., Hlisníková, H., Beronius, A., & Svingen, T. (2024). Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance. Reproductive Toxicology, 128, 108662. https://doi.org/10.1016/j.reprotox.2024.108662

Laier P, Metzdorff SB, Borch J, Hagen ML, Hass U, Christiansen S, Axelstad M, Kledal T, Dalgaard M, McKinnell C, Brokken LJ, & Vinggaard AM. (2006). Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz. Toxicology and Applied Pharmacology, 213(2), 160–171. https://doi.org/10.1016/j.taap.2005.10.013

Li C, Zou C, Yan H, Li Z, Li Y, Pan P, Ma F, Yu Y, Wang Y, Wen Z, & Ge RS. (2021). Perfluorotridecanoic acid inhibits fetal Leydig cell differentiation after in utero exposure in rats via increasing oxidative stress and autophagy. Environmental Toxicology, 36(6), 1206–1216. https://doi.org/10.1002/tox.23119

Liu C, Xu X, Zhang Y, Li W, & Huo X. (2016). Associations between maternal phenolic exposure and cord sex hormones in male newborns. Human Reproduction (Oxford, England), 31(3), 648–656. https://doi.org/10.1093/humrep/dev327

Lu, C.-L., Wen, H.-J., Chen, M.-L., Sun, C.-W., Hsieh, C.-J., Wu, M.-T., Wang, S.-L., & TMICS study grp. (2024). Prenatal phthalate exposure and sex steroid hormones in newborns: Taiwan Maternal and Infant Cohort Study. PLOS ONE, 19(3). https://doi.org/10.1371/journal.pone.0297631

Mably TA, Moore RW, & Peterson RE. (1992). In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Effects on androgenic status. Toxicology and Applied Pharmacology, 114(1), 97–107. https://doi.org/10.1016/0041-008x(92)90101-w

Murashima, A., Kishigami, S., Thomson, A., & Yamada, G. (2015). Androgens and mammalian male reproductive tract development. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1849(2), 163–170. https://doi.org/10.1016/j.bbagrm.2014.05.020

Pan P, Ma F, Wu K, Yu Y, Li Y, Li Z, Chen X, Huang T, Wang Y, & Ge RS. (2020). Maternal exposure to zearalenone in masculinization window affects the fetal Leydig cell development in rat male fetus. Environmental Pollution (Barking, Essex : 1987), 263, 114357. https://doi.org/10.1016/j.envpol.2020.114357

Schwartz CL, Christiansen S, Vinggaard AM, 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 RM. (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 KK, Acerini CL, & Hughes IA. (2016). Anogenital distance as a marker of androgen exposure in humans. Andrology, 4(4), 616–625. https://doi.org/10.1111/andr.12156

Trost, L. W., & Mulhall, J. P. (2016). Challenges in Testosterone Measurement, Data Interpretation, and Methodological Appraisal of Interventional Trials. The Journal of Sexual Medicine, 13(7), 1029–1046. https://doi.org/10.1016/j.jsxm.2016.04.068

Vesper, H. W., Wang, Y., Vidal, M., Botelho, J. C., & Caudill, S. P. (2015). Serum Total Testosterone Concentrations in the US Household Population from the NHANES 2011-2012 Study Population. Clinical Chemisty, 61(12), 1495–1504. https://doi.org/10.1373/clinchem.2015.245969

Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, Jacobsen H, Dalgaard M, Nellemann C, & Hass U. (2005). Perinatal exposure to the fungicide prochloraz feminizes the male rat offspring. Toxicological Sciences : An Official Journal of the Society of Toxicology, 85(2), 886–897. https://doi.org/doi.org/10.1093/toxsci/kfi150

Welsh M, Suzuki H, & Yamada G. (2014). The masculinization programming window. Endocrine Development, 27, 17–27. https://doi.org/10.1159/000363609

Zarean M, Keikha M, Feizi A, Kazemitabaee M, & Kelishadi R. (2019). The role of exposure to phthalates in variations of anogenital distance: A systematic review and meta-analysis. Environmental Pollution (Barking, Essex : 1987), 247, 172–179. https://doi.org/10.1016/j.envpol.2019.01.026