Relationship: 3448: Decrease, intratesticular testosterone leads to Decrease, circulating testosterone levels

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic applicability

The KER is assessed applicable to mammals, as testicular testosterone synthesis is common for all mammals. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates.

Sex applicability

This KER is only applicable to males, as testes are only found in males.

Life stage applicability

This KER is applicable to all life stages.  Once formed, the testes produce and secrete testosterone during fetal development and throughout postnatal life, although testosterone levels do vary between life stages (Vesper et al., 2015).

Key Event Relationship Description

This KE describes a decrease in intratesticular testosterone production leading to a decrease in circulating levels of testosterone. Intratesticular testosterone can be measured in whole testicular tissue samples by testing ex vivo testicular testosterone production, and circulating testosterone is measured in plasma or serum. In males, the testes produce and secrete the majority of the circulating testosterone, with only a small contribution from the adrenal gland (Naamneh Elzenaty et al., 2022). In mammals, intratesticular testosterone levels are 30- to 100-fold higher than serum testosterone levels (Coviello et al., 2004; McLachlan et al., 2002; Turner et al., 1984). Reducing testicular testosterone will consequently lead to a reduction in circulating levels as well.

Evidence Supporting this KER

The biological plausibility for this KER is considered high. The testes are the primary testosterone-producing organs in male mammals and the main contributors to the circulating testosterone levels in males (Naamneh Elzenaty et al., 2022). A decrease in intratesticular testosterone will therefore lead to a decrease in secretion of testosterone and consequently lower circulating levels of testosterone.  

The empirical evidence for this KER is overall judged as high.

In vivo toxicity studies in rats and mice have shown that exposure to substances that lowers intratesticular testosterone also lowers circulating testosterone levels. This includes in utero exposure and measurements in fetal males (Borch J et al., 2004; Vinggaard AM et al., 2005) as well as exposure and measurements postnatally in male rodents (Hou X et al., 2020; Ji et al., 2010; Jiang XP et al., 2017)

Supporting this evidence are castration studies in male rats and monkeys, showing a marked reduction in circulating testosterone levels when removing the testes (Gomes & Jain, 1976; Perachio et al., 1977).

Lastly, in humans, males with hypogonadism or gonadal dysgenesis present with lower circulating testosterone levels (Hirose Y et al., 2007; Jones LW et al., 1970).

Dose concordance

In vivo toxicity studies support dose concordance for this KER, as exemplified below.

In pre-pubertal/pubertal male rats, chlorocholine chloride exposure (postnatal day (PND) 23-60) in three doses reduced both intratesticular and serum testosterone levels at PND60 at all doses tested (Hou X et al., 2020).

Perinatal exposure (gestational day (GD) 10-birth) of male mice to diethylhexyl phthalate (DEHP) in three doses (100, 500, and 1000 mg/kg bw/day) reduced intratesticular testosterone at 500 and 1000 mg/kg bw/day at PND1, while only 1000 mg/kg bw/day reduced serum levels of testosterone, although this was measured later, at PND56 (Xie Q et al., 2024)

In utero exposure (GD7-21) of male rats to DEHP in doses of 300 or 750 mg/kg bw/day reduced intratesticular testosterone levels at GD21, while only the high dose also reduced plasma testosterone levels (Borch J et al., 2004).

Temporal concordance

In vivo toxicity studies moderately support temporal concordance for this KER, as exemplified below.

Several studies show that a decrease in intratesticular and circulating testosterone can be measured at the same time point (Borch J et al., 2004; Hou X et al., 2020; Jiang XP et al., 2017; Vinggaard AM et al., 2005).

In utero exposure of male mice to DEHP from GD10 to birth reduced intratesticular testosterone levels at PND1 with LOAEL 500 mg/kg bw/day, and when measured at PND56, circulating testosterone levels were decreased, but with LOAEL 1000 mg/kg bw/day (Xie Q et al., 2024).

In Fisher JS et al., 2003, exposure of male rats from GD13-21 to 500 mg/kg bw/day dibutyl phthalate reduced intratesticular testosterone by ~90% (measured at GD19). When analyzing circulating testosterone levels at PND4, 10, 15, 25, and 90, only the testosterone levels on PND25 were decreased.

One study report conflicting results on the temporal concordance of this KER (Caceres et al., 2023). Here, male rats were exposed for 20 weeks from PND60 to a mixture of the phytoestrogens genistein and daidzein (combined dose of either 29 or 290 mg/kg bw/day). Intratesticular testosterone was measured every 4 weeks, while serum levels of testosterone were measured every second week. While the mixture caused a reduction of serum testosterone after 2 weeks of exposure, a reduction in intratesticular testosterone was not measured until after 8 weeks. The discrepancy might be explained by the multiple mechanisms of action of the phytoestrogens, as they, besides affecting testicular testosterone synthesis, may also influence peripheral aromatization of testosterone to estrogens (van Duursen et al., 2011).

Incidence concordance

Incidence concordance can not be evaluated for this KER.

There are examples of in vivo studies, in which stressors exposure have caused a reduction in intratesticular testosterone levels without a reduction in circulating testosterone levels.

Quantitative Understanding of the Linkage

The time-scale for this KER is likely minutes or hours, as testosterone is secreted into the blood from the testes after synthesis. In vivo, a decrease in intratesticular and circulating testosterone can be measured at the same time, both in fetal and postnatal studies (Borch J et al., 2004; Hou X et al., 2020; Jiang XP et al., 2017; Vinggaard AM et al., 2005). Ex vivo, chemically-induced reduction in testicular production of testosterone can be measured in culture media after 3 hours incubation (earlier time points were not measured) (Wilson et al., 2009).

Testosterone is a part of the hypothalamic-pituitary-gonadal (HPG) axis, which controls testosterone synthesis in puberty and adulthood. In this axis, gonatropin-releasing hormone (GnRH) is released from the hypothalamus and stimulates release of luteinizing hormone (LH) from the pituitary. LH acts on the testes to produce and secrete testosterone. Elevated circulating testosterone levels exerts negative feedback on the HPG axis (decreasing GnRH secretion) to keep testosterone levels in balance (Tilbrook & Clarke, 2001).

Importantly, there are species-specific differences in when the HPG axis is functional during development. In the mouse, fetal testosterone synthesis is independent of pituitary LH (O’Shaughnessy et al., 1998), whereas in humans, human chorionic gonadotropin (hCG) act similarly to LH and appear to be critical in stimulating testosterone synthesis in the fetal testis (Huhtaniemi, 2025).

References

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

Caceres, S., Crespo, B., Alonso-Diez, A., De Andrés, P. J., Millan, P., Silván, G., Illera, M. J., & Illera, J. C. (2023). Long-Term Exposure to Isoflavones Alters the Hormonal Steroid Homeostasis-Impairing Reproductive Function in Adult Male Wistar Rats. Nutrients, 15(5), 1261. https://doi.org/10.3390/nu15051261

Coviello, A. D., Bremner, W. J., Matsumoto, A. M., Herbst, K. L., Amory, J. K., Anawalt, B. D., Yan, X., Brown, T. R., Wright, W. W., Zirkin, B. R., & Jarow, J. P. (2004). Intratesticular Testosterone Concentrations Comparable With Serum Levels Are Not Sufficient to Maintain Normal Sperm Production in Men Receiving a Hormonal Contraceptive Regimen. Journal of Andrology, 25(6), 931–938. https://doi.org/10.1002/j.1939-4640.2004.tb03164.x

Fisher JS, Macpherson S, Marchetti N, & Sharpe RM. (2003). Human “testicular dysgenesis syndrome”: A possible model using in-utero exposure of the rat to dibutyl phthalate. Human Reproduction (Oxford, England), 18(7), 1383–1394. https://doi.org/10.1093/humrep/deg273

Gomes, W. R., & Jain, S. K. (1976). Effect of unilateral and bilateral castration and cryptorchidism on serum gonadotrophins in the rat. The Journal of Endocrinology, 68(02), 191–196. https://doi.org/10.1677/joe.0.0680191

Hirose Y, Sasa M, Bando Y, Hirose T, Morimoto T, Kurokawa Y, Nagao T, & Tangoku A. (2007). Bilateral male breast cancer with male potential hypogonadism. World Journal of Surgical Oncology, 5, 60. https://doi.org/10.1186/1477-7819-5-60

Hou X, Hu H, Xiagedeer B, Wang P, Kang C, Zhang Q, Meng Q, & Hao W. (2020). Effects of chlorocholine chloride on pubertal development and reproductive functions in male rats. Toxicology Letters, 319, 1–10. https://doi.org/10.1016/j.toxlet.2019.10.024

Huhtaniemi, I. T. (2025). Luteinizing hormone receptor knockout mouse: What has it taught us? Andrology, andr.70000. https://doi.org/10.1111/andr.70000

Ji, Y.-L., Wang, H., Liu, P., Wang, Q., Zhao, X.-F., Meng, X.-H., Yu, T., Zhang, H., Zhang, C., Zhang, Y., & Xu, D.-X. (2010). Pubertal cadmium exposure impairs testicular development and spermatogenesis via disrupting testicular testosterone synthesis in adult mice. Reproductive Toxicology, 29(2), 176–183. https://doi.org/10.1016/j.reprotox.2009.10.014

Jiang XP, Tang JY, Xu Z, Han P, Qin ZQ, Yang CD, Wang SQ, Tang M, Wang W, Qin C, Xu Y, Shen BX, Zhou WM, & Zhang W. (2017). Sulforaphane attenuates di-N-butylphthalate-induced reproductive damage in pubertal mice: Involvement of the Nrf2-antioxidant system. Environmental Toxicology, 32(7), 1908–1917. https://doi.org/10.1002/tox.22413

Jones LW, Isaacs H Jr, Edelbrock H, & Donnell GN. (1970). Reifenstein’s syndrome: Hereditary familial hypogonadism with hypospadias and gynecomastia. The Journal of Urology, 104(4), 608–611. https://doi.org/10.1016/s0022-5347(17)61793-2

McLachlan, R. I., O’Donnell, L., Stanton, P. G., Balourdos, G., Frydenberg, M., de Kretser, D. M., & Robertson, D. M. (2002). Effects of Testosterone Plus Medroxyprogesterone Acetate on Semen Quality, Reproductive Hormones, and Germ Cell Populations in Normal Young Men. The Journal of Clinical Endocrinology & Metabolism, 87(2), 546–556. https://doi.org/10.1210/jcem.87.2.8231

Naamneh Elzenaty, R., Du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. Best Practice & Research Clinical Endocrinology & Metabolism, 36(4), 101665. https://doi.org/10.1016/j.beem.2022.101665

O’Shaughnessy, P. J., Baker, P., Sohnius, U., Haavisto, A.-M., Charlton, H. M., & Huhtaniemi, I. (1998). Fetal Development of Leydig Cell Activity in the Mouse Is Independent of Pituitary Gonadotroph Function*. Endocrinology, 139(3), 1141–1146. https://doi.org/10.1210/endo.139.3.5788

Perachio, A. A., Alexander, M., Marr, L. D., & Collins, D. C. (1977). Diurnal variations of serum testosterone levels in intact and gonadectomized male and female rhesus monkeys. Steroids, 29(1), 21–33. https://doi.org/10.1016/0039-128X(77)90106-4

Tilbrook, A. J., & Clarke, I. J. (2001). Negative Feedback Regulation of the Secretion and Actions of Gonadotropin-Releasing Hormone in Males. Biology of Reproduction, 64(3), 735–742. https://doi.org/10.1095/biolreprod64.3.735

Turner, T. T., Jones, C. E., Howards, S. S., Ewing, L. L., Zegeye, B., & Gunsalus, G. L. (1984). On the androgen microenvironment of maturing spermatozoa. Endocrinology, 115(5), 1925–1932. https://doi.org/10.1210/endo-115-5-1925

van Duursen, M. B. M., Nijmeijer, S. M., de Morree, E. S., de Jong, P. Chr., & van den Berg, M. (2011). Genistein induces breast cancer-associated aromatase and stimulates estrogen-dependent tumor cell growth in in vitro breast cancer model. Toxicology, 289(2), 67–73. https://doi.org/10.1016/j.tox.2011.07.005

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

Wilson, V. S., Lambright, C. R., Furr, J. R., Howdeshell, K. L., & Gray, L. E., Jr. (2009). The herbicide linuron reduces testosterone production from the fetal rat testis during both in utero and in vitro exposures. TOXICOLOGY LETTERS, 186(2), 73–77. https://doi.org/10.1016/j.toxlet.2008.12.017

Xie Q, Cao H, Liu H, Xia K, Gao Y, & Deng C. (2024). Prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression. Translational Andrology and Urology, 13(3), 369–382. https://doi.org/10.21037/tau-23-503

Relationship: 2131: Decrease, circulating testosterone levels leads to Decrease, AR activation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic applicability

KER2131 is assessed applicable to mammals, as T and AR activation are known to be related in mammals. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.

Sex applicability

KER2131 is assessed applicable to both sexes, as T activates AR in both males and females.

Life-stage applicability

KER2131 is considered applicable to developmental and adult life stages, as T-mediated AR activation is relevant from the AR is expressed.

Key Event Relationship Description

This key event relationship links decreased testosterone (T) levels to decreased androgen receptor (AR) activation. T is an endogenous steroid hormone important for, amongst other things, reproductive organ development and growth as well as muscle mass and spermatogenesis (Marks, 2004).T is, together with dihydrotestosterone (DHT), a primary ligand for the AR in mammals (Schuppe et al., 2020). Besides its genomic actions, the AR can also mediate rapid, non-genomic second messenger signaling (Davey & Grossmann, 2016). When T levels are reduced, less substrate is available for the AR, and hence, AR activation is decreased (Gao et al., 2005).

Evidence Supporting this KER

The biological plausibility for this KER is considered high

AR activation is dependent on ligand binding (though a few cases of ligand-independent AR activation has been shown, see uncertainties and inconsistencies). T is a primary ligand for the AR, and when T levels are decreased there is less substrate for the AR, and hence, AR activation is decreased. In the male, T is primarily synthesized by the testes, and in some target tissues T is irreversibly metabolized to the more potent metabolite DHT. T and DHT both bind to the AR, but DHT has a higher binding affinity (Gao et al., 2005). The lower binding affinity of T compared to DHT is due to the faster dissociation rate of T from the full-length AR, as T has less effective FXXLF motif binding to AF2 (Askew et al., 2007). Binding of T or DHT has different effects in different tissues. E.g. in the developing male, T is required for development of the internal sex organs (epididymis, vas deferens and the seminal vesicles), whereas DHT is crucial for development of the external sex organs (Keller et al., 1996). In the adult male, androgen action in the reproductive tissues is DHT dependent, whereas action in muscle and bone is DHT independent (Gao et al., 2005). In patients with male androgen deficiency syndrome (AIS), clinically low levels of T leads to reduced AR activation (either due to low T or DHT in target tissue), which manifests as both androgenic related symptoms (such as incomplete or delayed sexual development, loss of body hair, small or shrinking testes, low or zero sperm count) as well as anabolic related symptoms (such as height loss, low trauma fracture, low bone mineral density, reduced muscle bulk and strength, increased body fat). All symptoms can be counteracted by treatment with T, which acts directly on the AR receptor in anabolic tissue (Bhasin et al., 2010). Similarly, removal of the testicles in weanling rats results in a feminized body composition and muscle metabolism, which is reversed by administration of T (Krotkiewski et al., 1980). As this demonstrates, the consequences of low T regarding AR activation will depend on tissue, life stage, species etc.

The empirical evidence for this KER is considered high

Dose concordance

There is a positive dose-response relationship between increasing concentrations of T and AR activation (U.S. EPA., 2023).

Other evidence

• In male patients with androgen deficiency, treatment with T counteracts anabolic (DHT independent) related symptoms such as height loss, low trauma fracture, low bone mineral density, reduced muscle bulk and strength, increased body fat (Bhasin et al., 2010; Katznelson et al., 1996).
• Removal of the testicles in weanling rats result in a feminized body composition and muscle metabolism, which is reversed by administration of T (Krotkiewski et al., 1980).

Ligand-independent actions of the AR have been identified. To what extent and of which biological significance is not well defined (Bennesch & Picard, 2015).

Quantitative Understanding of the Linkage

There is a positive dose-response relationship between increasing concentrations of T and AR activation (U.S. EPA., 2023). However, there is not enough data, or overview of the data, to define a quantitative linkage in vivo, and such a relationship will differ between biological systems (species, tissue, cell type).

AR and promoter interactions occur within 15 minutes of ligand binding, and RNA polymerase II and coactivator recruitment are then proposed to occur transiently with cycles of approximately 90 minutes (Kang et al., 2002).

Androgens can upregulate and downregulate AR expression (Lee & Chang, 2003).

References

Askew, E. B., Gampe, R. T., Stanley, T. B., Faggart, J. L., & Wilson, E. M. (2007). Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone. Journal of Biological Chemistry, 282(35), 25801–25816. https://doi.org/10.1074/jbc.M703268200

Bennesch, M. A., & Picard, D. (2015). Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors. Molecular Endocrinology, 29(3), 349–363. https://doi.org/10.1210/me.2014-1315

Bhasin, S., Cunningham, G. R., Hayes, F. J., Matsumoto, A. M., Snyder, P. J., Swerdloff, R. S., & Montori, V. M. (2010). Testosterone Therapy in Men with Androgen Deficiency Syndromes: An Endocrine Society Clinical Practice Guideline. The Journal of Clinical Endocrinology & Metabolism, 95(6), 2536–2559. https://doi.org/10.1210/jc.2009-2354

Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. The Clinical Biochemist. Reviews, 37(1), 3–15. http://www.ncbi.nlm.nih.gov/pubmed/27057074

Gao, W., Bohl, C. E., & Dalton, J. T. (2005). Chemistry and Structural Biology of Androgen Receptor. Chemical Reviews, 105(9), 3352–3370. https://doi.org/10.1021/cr020456u

Kang, Z., Pirskanen, A., Jänne, O. A., & Palvimo, J. J. (2002). Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex. Journal of Biological Chemistry, 277(50), 48366–48371. https://doi.org/10.1074/jbc.M209074200

Katznelson, L., Finkelstein, J. S., Schoenfeld, D. A., Rosenthal, D. I., Anderson, E. J., & Klibanski, A. (1996). Increase in bone density and lean body mass during testosterone administration in men with acquired hypogonadism. The Journal of Clinical Endocrinology & Metabolism, 81(12), 4358–4365. https://doi.org/10.1210/jcem.81.12.8954042

Keller, E. T., Ershler, W. B., & Chang, Chawnshang. (1996). The androgen receptor: A mediator of diverse responses. Frontiers in Bioscience, 1(4), 59–71. https://doi.org/10.2741/A116

Krotkiewski, M., Kral, J. G., & Karlsson, J. (1980). Effects of castration and testosterone substitution on body composition and muscle metabolism in rats. Acta Physiologica Scandinavica, 109(3), 233–237. https://doi.org/10.1111/j.1748-1716.1980.tb06592.x

Lee, D. K., & Chang, C. (2003). Expression and Degradation of Androgen Receptor: Mechanism and Clinical Implication. The Journal of Clinical Endocrinology & Metabolism, 88(9), 4043–4054. https://doi.org/10.1210/jc.2003-030261

Marks, L. S. (2004). 5alpha-reductase: history and clinical importance. Reviews in Urology, 6 Suppl 9(Suppl 9), S11-21. http://www.ncbi.nlm.nih.gov/pubmed/16985920

Schuppe, E. R., Miles, M. C., and Fuxjager, M. J. (2020). Evolution of the androgen receptor: Perspectives from human health to dancing birds. Mol. Cell. Endocrinol. 499, 110577. doi:10.1016/J.MCE.2019.110577.

U.S. EPA. (2023). ToxCast & Tox21 AR agonism of testosterone. Retrieved from Https://Www.Epa.Gov/Chemical-Research/Toxicity-Forecaster-Toxcasttm-Data June 23, 2023. Data Released October 2018.

Relationship: 2124: Decrease, AR activation leads to Altered, Transcription of genes by the AR

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

This KER is applicable for both sexes, across developmental stages into adulthood, in numerous cells and tissues and across mammalian taxa. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.

Key Event Relationship Description

The androgen receptor (AR) is a ligand-dependent nuclear transcription factor that upon activation translocates to the nucleus, dimerizes, and binds androgen response elements (AREs) to modulate transcription of target genes (Lamont and Tindall, 2010, Roy et al. 2001). Decreased activation of the AR affects its transcription factor activity, therefore leading to altered AR-target gene expression. This KER refers to decreased AR activation and altered gene expression occurring in complex systems, such as in vivo and the specific effect on transcription of AR target genes will depend on species, life stage, tissue, cell type etc.

Evidence Supporting this KER

The biological plausibility for this KER is considered high

The AR is a ligand-activated transcription factor part of the steroid hormone nuclear receptor family. Non-activated AR is found in the cytoplasm as a multiprotein complex with heat-shock proteins, immunophilins and, other chaperones (Roy et al. 2001). Upon activation through ligand binding, the AR dissociates from the protein complex, translocates to the nucleus and homodimerizes. Facilitated by co-regulators, AR can bind to DNA regions containing AREs and initiate transcription of target genes, that thus will be different in e.g. different tissues, life-stages, species etc.

Through mapping of AREs and ChIP sequencing studies, several AR target genes have been identified, mainly studied in prostate cells (Jin, Kim, and Yu 2013). Different co-regulators and ligands lead to altered expression of different sets of genes (Jin et al. 2013; Kanno et al. 2022). Alternative splicing of the AR can lead to different AR variants that also affects which genes are transcribed (Jin et al. 2013).

Apart from this canonical signaling pathway, the AR can suppress gene expression, indirectly regulate miRNA transcription, and have non-genomic effects by rapid activation of second messenger pathways in either presence or absence of a ligand (Jin et al. 2013).

The empirical evidence for this KER is considered high

In humans, altered gene expression profiling in individuals with androgen insensitivity syndrome (AIS) can provide supporting empirical evidence (Holterhus et al. 2003; Peng et al. 2021). In rodent AR knockout (KO) models, gene expression profiling studies and gene-targeted approaches have provided information on differentially expressed genes in several organ systems including male and female reproductive, endocrine, muscular, cardiovascular and nervous systems (Denolet et al. 2006; Fan et al. 2005; Holterhus et al. 2003; Ikeda et al. 2005; Karlsson et al. 2016; MacLean et al. 2008; Rana et al. 2011; Russell et al. 2012; Shiina et al. 2006; Wang et al. 2006; Welsh et al. 2012; Willems et al. 2010; Yu et al. 2008, 2012; Zhang et al. 2006; Zhou et al. 2011).

Exposure to known antiandrogens has been shown to alter transcriptional profiles, for example of neonatal pig ovaries (Knapczyk-Stwora et al. 2019).

Dose concordance has also been observed for instance in zebrafish embryos; a dose of 50 µg/L of the AR antagonist flutamide resulted in 674 differentially expressed genes at 96 h post fertilization whereas 500 µg/L flutamide resulted in 2871 differentially expressed genes (Ayobahan et al., 2023).

AR action has been reported to occur also without ligand binding. However, not much is known about the extent and biological implications of such non-canonical, ligand-independent AR activation (Bennesch and Picard 2015).

Quantitative Understanding of the Linkage

There is not enough data to define a quantitative relationship between AR activation and alteration of AR target gene transcription, and such a relationship will differ between biological systems (species, tissue, cell type, life stage etc).

AR and promoter interactions occur within 15 minutes of ligand binding, RNA polymerase II and coactivator recruitment are proposed to occur transiently with cycles of approximately 90 minutes in LNCaP cells (Kang et al. 2002). RNA polymerase II elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb/min (Maiuri et al. 2011). Therefore, depending on the cell type and the half-life of the AR target gene transcripts, changes are to be expected within hours.

AR has been hypothesized to auto-regulate its mRNA and protein levels (Mora and Mahesh 1999).

References

Ayobahan, S. U., Alvincz, J., Reinwald, H., Strompen, J., Salinas, G., Schäfers, C., et al. (2023). Comprehensive identification of gene expression fingerprints and biomarkers of sexual endocrine disruption in zebrafish embryo. Ecotoxicol. Environ. Saf. 250, 114514. doi:10.1016/J.ECOENV.2023.114514.

Bennesch, Marcela A., and Didier Picard. 2015. “Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors.” Molecular Endocrinology 29(3):349–63.

Chamberlain, Nancy L., Erika D. Driverand, and Roger L. Miesfeldi. 1994. The Length and Location of CAG Trinucleotide Repeats in the Androgen Receptor N-Terminal Domain Affect Transactivation Function. Vol. 22.

Denolet, Evi, Karel De Gendt, Joke Allemeersch, Kristof Engelen, Kathleen Marchal, Paul Van Hummelen, Karen A. L. Tan, Richard M. Sharpe, Philippa T. K. Saunders, Johannes V. Swinnen, and Guido Verhoeven. 2006. “The Effect of a Sertoli Cell-Selective Knockout of the Androgen Receptor on Testicular Gene Expression in Prepubertal Mice.” Molecular Endocrinology 20(2):321–34. doi: 10.1210/me.2005-0113.

Fan, Wuqiang, Toshihiko Yanase, Masatoshi Nomura, Taijiro Okabe, Kiminobu Goto, Takashi Sato, Hirotaka Kawano, Shigeaki Kato, and Hajime Nawata. 2005. Androgen Receptor Null Male Mice Develop Late-Onset Obesity Caused by Decreased Energy Expenditure and Lipolytic Activity but Show Normal Insulin Sensitivity With High Adiponectin Secretion. Vol. 54.

Holterhus, Paul-Martin, Olaf Hiort, Janos Demeter, Patrick O. Brown, and James D. Brooks. 2003. Differential Gene-Expression Patterns in Genital Fibroblasts of Normal Males and 46,XY Females with Androgen Insensitivity Syndrome: Evidence for Early Programming Involving the Androgen Receptor. Vol. 4.

Ikeda, Yasumasa, Ken Ichi Aihara, Takashi Sato, Masashi Akaike, Masanori Yoshizumi, Yuki Suzaki, Yuki Izawa, Mitsunori Fujimura, Shunji Hashizume, Midori Kato, Shusuke Yagi, Toshiaki Tamaki, Hirotaka Kawano, Takahiro Matsumoto, Hiroyuki Azuma, Shigeaki Kato, and Toshio Matsumoto. 2005. “Androgen Receptor Gene Knockout Male Mice Exhibit Impaired Cardiac Growth and Exacerbation of Angiotensin II-Induced Cardiac Fibrosis.” Journal of Biological Chemistry 280(33):29661–66. doi: 10.1074/jbc.M411694200.

Jin, Hong Jian, Jung Kim, and Jindan Yu. 2013. “Androgen Receptor Genomic Regulation.” Translational Andrology and Urology 2(3):158–77.

Kang, Zhigang, Asta Pirskanen, Olli A. Jänne, and Jorma J. Palvimo. 2002. “Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex.” Journal of Biological Chemistry 277(50):48366–71. doi: 10.1074/jbc.M209074200.

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Karlsson, Sara A., Erik Studer, Petronella Kettunen, and Lars Westberg. 2016. “Neural Androgen Receptors Modulate Gene Expression and Social Recognition but Not Social Investigation.” Frontiers in Behavioral Neuroscience 10(MAR). doi: 10.3389/fnbeh.2016.00041.

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Relationship: 3488: Decrease, intratesticular testosterone leads to Hypospadias

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic applicability

The development and differentiation of the penis is driven by androgen hormones, mainly produced by fetal testes, in all mammals. It is therefore biologically plausible that this KER is applicable to all mammals (Murashima et al., 2015). The empirical evidence in this KER provides support that reduced intratesticular testosterone levels in fetal life can cause hypospadias in rats. Studies in humans with gonadal dysgenesis and concurrent hypospadias support this KER’s applicability to humans (Boehmer et al., 2001; Crone et al., 2002).

Sex applicability

This KER is applicable to males, where the testes are the primary sex organ.

Life stage applicability

The genital tubercle is programmed by androgen hormones in the masculinization programming window (gestational days (GD) 16-20 in rats, and gestational weeks (GW) 8-14 in humans ), when the testes produce high levels of testosterone (Sharpe, 2020; Welsh et al., 2014). The genital tubercle starts differentiating in fetal life, and in humans the penis is fully formed at birth, where hypospadias is usually diagnosed (Yu et al., 2019). In rats and mice, penis development continues postnatally for around 20-25 days, and hypospadias is optimally diagnosed after this timepoint, although it may also be observed earlier (Schlomer et al., 2013; Sinclair et al., 2017).

Key Event Relationship Description

This non-adjacent KER describes a fetal decrease in testis testosterone leading to hypospadias in male offspring. In this KER, intratesticular testosterone levels can both be measured in whole testes homogenates or by measuring ex vivo testosterone production from cultured testes.

In male mammals, the testes differentiate in early fetal life and begin steroidogenesis to synthesize testosterone. Testosterone is secreted from the fetal testes for initiation of differentiation of the male reproductive tissues. Testosterone acts at the androgen receptor (AR) or is converted by 5α-reductase to the more potent androgen dihydrotestosterone (DHT). Activation of AR in the bipotential genital tubercle starts differentiation into a penis. While penis differentiation is a longer process, programming of the genital tubercle is largely constrained to a fixed period (GD 16-20 in rats, GW 8-14 in humans), when testicular testosterone production is high (Sharpe, 2020; Welsh et al., 2014). Failure of proper penis differentiation can cause genital malformations, of which the most common is hypospadias, where the urethral opening is on the underside of the penis.

A decrease in intratesticular testosterone levels may therefore lead to hypospadias in male offspring.

Evidence Supporting this KER

The biological plausibility for this KER is judged to be high given the canonical biological knowledge on normal reproductive development.

Differentiation of the penis is programmed during fetal development. Once the testes have formed around GW8 in humans and GD16 in rats, they synthesize testosterone through the steroidogenesis pathway (Murashima et al., 2015). Although the adrenal glands may also produce testosterone, the testes are the main site of testosterone production (Naamneh Elzenaty et al., 2022).  Testosterone is secreted from the testes and is transported to the peripheral tissues, including the genital tubercle. Testosterone may act directly on the AR or be converted to the more potent androgen DHT.

The genital tubercle is the bipotential structure that upon hormonal cues differentiates to either penis or clitoris. Both human and rodent genital tubercles express AR (C. M. Amato & Yao, 2021; Baskin et al., 2020). Upon activation of AR, the genital tubercle differentiates to a penis by elongation and formation of a central urethra which terminates at the tip of the penis (C. Amato et al., 2022). The programming of the genital tubercle happens in the masculinization programming window (GD 16-20 in rats, GW 8-14 in humans) (Welsh et al., 2014), although elongation and growth of the penis is also programmed later, at least in rats (Welsh et al., 2008). Hypospadias is one of the most common genital malformations caused by disruptions to penis development (Baskin & Ebbers, 2006; Yu et al., 2019).

Given the dependency of testosterone for penis differentiation, either through direct AR activation or conversion to DHT, it is plausible that a decrease in intratesticular testosterone will cause hypospadias.

The empirical evidence from studies in animals for this KER is overall judged as moderate

From the data collection, three data sets were extracted. The data sets included different stressors causing reduced fetal levels of testosterone, all in rats (table 2 and appendix 2, 59p4izmm69_KER_3488_appendix_2.pdf). All studies showed concurrent hypospadias in the male offspring.

Table 2 Empirical evidence for KER 3488 LOAEL: Lowest observed adverse effect level; see appendix 2 for specifications: 59p4izmm69_KER_3488_appendix_2.pdf

Supporting human studies

Supporting the empirical evidence are human cases of patients with less testicular tissue (i.e. partial gonadal dysgenesis) which can cause severe hypospadias (Boehmer et al., 2001; Crone et al., 2002).

Dose concordance

One study informs and supports dose concordance for this KER. In this study, diisocytol phthalate caused reduced ex vivo testosterone production at a dose of 0.1 mg/kg bw/day, while hypospadias was observed in male offspring at 1 mg/kg bw/day (Saillenfait et al., 2013).

Temporal concordance

Empirical evidence indirectly supports temporal concordance. In all studies, the exposure window was prenatal, and low testosterone levels were detected at GD18-22, while hypospadias was assessed in adult rats. There are, however, no assessments of hypospadias at earlier timepoints to establish when the phenotype became visible.

Incidence concordance

Incidence concordance cannot be directly informed from the empirical evidence, because testosterone levels are reported as means of all values and is a continuous variable. However, given that hypospadias was not registered in all males in any of the studies, this could suggest a higher incidence of lower testosterone levels than the incidence of hypospadias.

In one study (Drake et al., 2009), testosterone levels were only reduced when performing statistical analysis on individual values and not on litter means. Hypospadias was observed in 30% of males. The difference in statistical significance between litter means and individual values is an uncertainty to this study.

In (Saillenfait et al., 2013), intratesticular testosterone was only measured in ex vivo testes cultures, which are assumed to be a good proxy for intratesticular testosterone levels, although it should be kept in mind as an uncertainty.

Another uncertainty for this KER from the literature is the observation that humans with 5α-reductase deficiency have hypospadias due to low DHT levels despite normal or higher testosterone levels (Mendonca et al., 1996). This indicates that the effects of low testosterone may be more through reduced conversion to DHT than due to a direct loss of testosterone action on AR. To this, there is also the existence of a “backdoor pathway” to DHT in humans. This pathway in peripheral tissues (i.e. not testes) can circumvent testosterone as a precursor for DHT by synthesis of DHT is from reduction of androsterone by 17β-HSD (Miller & Auchus, 2019). This would create the possibility that testosterone is not required for DHT production and ultimately AR activation.

Quantitative Understanding of the Linkage

The quantitative understanding of this KER is low.

A model for phthalate-induced malformations has been developed which aims to predict the frequency of hypospadias related to a phthalate’s reduction in ex vivo testosterone production. The model predicted that a 60% reduction in testosterone levels would induce hypospadias, although the predictivity of this model was not good when tested for one phthalate (Earl Gray et al., 2024).

The time-scale of this KER largely depends on species, but is likely weeks. In humans, the masculinization programming window is weeks long, while in rodents it is days (Sharpe, 2020; Welsh et al., 2014). Hypospadias is diagnosed at birth in humans (Yu et al., 2019) and can also be observed at birth in rodents, but as development of the penis continues after birth in rodents, hypospadias may be more optimally evaluated later in juvenile or adult male rats (Schlomer et al., 2013; Sinclair et al., 2017).

There are no known modulating factors for this KER

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

References

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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

Baskin, L., Cao, M., Sinclair, A., Li, Y., Overland, M., Isaacson, D., & Cunha, G. R. (2020). Androgen and estrogen receptor expression in the developing human penis and clitoris. Differentiation; Research in Biological Diversity, 111, 41–59. https://doi.org/10.1016/j.diff.2019.08.005

Baskin, L., & Ebbers, M. (2006). Hypospadias: Anatomy, etiology, and technique. Journal of Pediatric Surgery, 41(3), 463–472. https://doi.org/10.1016/j.jpedsurg.2005.11.059

Boehmer, A., Nijman, R., Lammers, B., de Coninck, S., Van Hemel, J., Themmen, A., Mureau, M., de Jong, F., Brinkmann, A., Niermeijer, M., & Drop, S. (2001). Etiological studies of severe or familial hypospadias. The Journal of Urology, 165(4), 1246–1254.

Crone, J., Amann, G., Gheradini, R., Kirchlechner, V., & Fékété, C. (2002). Management of 46, XY partial gonadal dysgenesis—Revisited. Wiener Klinische Wochenschrift, 114(12), 462–467.

Drake, A., van den Driesche, S., Scott, H., Hutchison, G., Seckl, J., & Sharpe, R. (2009). Glucocorticoids Amplify Dibutyl Phthalate-Induced Disruption of Testosterone Production and Male Reproductive Development. ENDOCRINOLOGY, 150(11), 5055–5064. https://doi.org/10.1210/en.2009-0700

Earl Gray, L. J., Lambright, C., Evans, N., Ford, J., & Conley, M. (2024). Using targeted fetal rat testis genomic and endocrine alterations to predict the effects of a phthalate mixture on the male reproductive tract. Current Research in Toxicology, 7, 100180. https://doi.org/10.1016/j.crtox.2024.100180

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

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

Naamneh Elzenaty, R., Du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. Best Practice & Research Clinical Endocrinology & Metabolism, 36(4), 101665. https://doi.org/10.1016/j.beem.2022.101665

Saillenfait, A., Sabaté, J., Robert, A., Cossec, B., Roudot, A., Denis, F., & Burgart, M. (2013). Adverse effects of diisooctyl phthalate on the male rat reproductive development following prenatal exposure. Reproductive Toxicology (Elmsford, N.Y.), 42, 192–202. https://doi.org/10.1016/j.reprotox.2013.09.004

Schlomer, B. J., Feretti, M., Rodriguez, E. J., Blaschko, S., Cunha, G., & Baskin, L. (2013). Sexual differentiation in the male and female mouse from days 0 to 21: A detailed and novel morphometric description. The Journal of Urology, 190(4 Suppl), 1610–1617. https://doi.org/10.1016/j.juro.2013.02.3198

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

Sinclair, A., Cao, M., Pask, A., Baskin, L., & Cunha, G. (2017). Flutamide-induced hypospadias in rats: A critical assessment. Differentiation; Research in Biological Diversity, 94, 37–57. https://doi.org/10.1016/j.diff.2016.12.001

van den Driesche, S., Shoker, S., Inglis, F., Palermo, C., Langsch, A., & Otter, R. (2020). Systematic comparison of the male reproductive tract in fetal and adult Wistar rats exposed to DBP and DINP in utero during the masculinisation programming window. Toxicology Letters, 335, 37–50. https://doi.org/10.1016/j.toxlet.2020.10.006

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

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

Yu, X., Nassar, N., Mastroiacovo, P., Canfield, M., Groisman, B., Bermejo-Sánchez, E., Ritvanen, A., Kiuru-Kuhlefelt, S., Benavides, A., Sipek, A., Pierini, A., Bianchi, F., Källén, K., Gatt, M., Morgan, M., Tucker, D., Canessa, M. A., Gajardo, R., Mutchinick, O. M., … Agopian, A. J. (2019). Hypospadias Prevalence and Trends in International Birth Defect Surveillance Systems, 1980-2010. European Urology, 76(4), 482–490. https://doi.org/10.1016/j.eururo.2019.06.027

Relationship: 3350: Decrease, circulating testosterone levels leads to Hypospadias

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic applicability

Sexual differentiation of the penis is an androgen-driven process in mammals, and it is therefore biologically plausible that this KER is applicable to all mammals (Murashima et al., 2015). The empirical evidence in this KER provides support that hypospadias in humans is associated with reduced circulating testosterone levels in early life.  The two in vivo studies included in the empirical evidence support the applicability to rats.

Sex applicability

The empirical evidence in this KER supports that reduced circulating testosterone is linked to hypospadias in males. Females do have circulating testosterone, but in much lower concentrations than males (Vesper et al., 2015). Moreover, the term hypospadias is mainly used for malformation of the male external genitalia.  

Life stage applicability

The genital tubercle is programmed by the surge in androgen hormones during the masculinization programming window is (gestational days (GD) 16-20 in rats, and gestational weeks (GW) 8-14 in humans ) (Sharpe, 2020; Welsh et al., 2014). In humans, the penis is fully formed at birth (Yu et al., 2019), while penis development continues postnatally around 20-25 days in rats and mice (Schlomer et al., 2013; Sinclair et al., 2017).

Key Event Relationship Description

This non-adjacent KER describes a decrease in circulating testosterone (often measured in serum or plasma) during the fetal male masculinization programming window leading to hypospadias in male offspring.

In male mammals, testosterone along with its more potent derivative dihydrotestosterone (DHT) drives 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 DHT (Murashima et al., 2015). Activation of AR in the genital tubercle directs its differentiation to a penis, and failure of this differentiation can lead to malformations, including hypospadias where the urethra terminates on the underside of the penis. The androgen programming of the genital tubercle is largely (but not fully) constrained to the masculinization programming window (GD 16-20 in rats, GW 8-14 in humans), when circulating testosterone levels are high (Sharpe, 2020; Welsh et al., 2014).

A decrease in circulating testosterone levels in the masculinization programming window can thus disrupt penis differentiation and cause hypospadias.

Evidence Supporting this KER

The biological plausibility for this KER is judged to be high given the canonical biological knowledge on normal reproductive development.

Sexual differentiation in males, the external genitalia, is initiated and programmed in fetal life. Once the testes have formed, they synthesize testosterone through the steroidogenesis pathway and secrete it into circulation. Testosterone is transported in the blood either as free testosterone or bound to albumin or sex-hormone binding globulin. 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.  In peripheral tissues, testosterone can be converted to the more potent androgen 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 (Murashima et al., 2015; Trost & Mulhall, 2016; Welsh et al., 2014).

The genital tubercle is the bipotential structure that upon hormonal cues differentiates to either penis or clitoris. Both human and rodent genital tubercles express AR (C. M. Amato & Yao, 2021; Baskin et al., 2020). Upon activation of AR, the genital tubercle differentiates to a penis by elongation and formation of a central urethra which terminates at the tip of the penis (C. Amato et al., 2022). The programming of the genital tubercle happens in the masculinization programming window (GD 16-20 in rats, GW 8-14 in humans) (Welsh et al., 2014), although elongation and growth of the penis is also programmed later, at least in rats (Welsh et al., 2008). Hypospadias is one of the most common genital malformations caused by disruptions to penis development (Baskin & Ebbers, 2006; Yu et al., 2019).

Given the dependency of testosterone for penis differentiation, either through direct AR activation or conversion to DHT, it is plausible that a decrease in circulating levels of testosterone will cause hypospadias.

The empirical evidence from studies in animals for this KER is judged as low overall.

From the data collection, two toxicity studies were extracted which reported a decrease in circulating testosterone levels in fetal male rats and measured hypospadias in male offspring. However, both studies were classified as not reliable (category 3). Due to the lack of any reliable studies for this KER, the evidence from the two unreliable data sets is included, although they are considered weak evidence for the KER.

In this first study (Li et al., 2017), rats were exposed to 0 or 750 mg/kg bw/day dibutylphthalate (DBP) from GD13-18. At GD19, serum testosterone and hypospadias were evaluated. At this stage, 43.6% of males exposed to DBP had hypospadias upon examination. In the hypospadias group, serum testosterone levels were 3.93 nmol/mL compared to 15.74 nmol/mL in control male rats. In exposed rats without hypospadias, serum testosterone levels were 5.89 nmol/mL. This study was categorized as unreliable, mainly due to the evaluation of hypospadias being on GD19, a timepoint at which the penis is not finished differentiating. This poses an uncertainty, in particular regarding the frequency of hypospadias reported in the exposure group.

The second study (Vo et al., 2009) was considered unreliable due to a too low reliability score (64.7) with some key information not reported. In this study, no major deficiencies were noted. Rats were exposed to 0, 10, 100, or 500 mg/kg bw/day diethylhexylphthalate (DEHP) from GD11-21. At GD21, plasma testosterone levels were reduced in males in the highest exposure group (0.53 ±0.16 ng/mL) compared to control males (1.56 ±0.84 ng/mL), but not in the other dose groups. Hypospadias was evaluated in male offspring at PND63, and hypospadias was registered in 23/100 male pups exposed in utero to 500 mg/kg bw/day. There was no hypospadias in the other groups.

Supporting human case studies

Supporting this KER are case studies of humans with hypospadias and a reduction in circulating testosterone levels.

Studies extracted from the literature search are summarized in table 2.

Table 2: Human case studies of hypospadias with reductions in circulating testosterone levels (measured postnatally).

Dose concordance

Dose concordance cannot be informed from the two in vivo studies, although the study (Vo et al., 2009) does not argue against dose concordance as the upstream and downstream events were measured at the same dose of DEHP.

Temporal concordance

The in vivo studies do not directly inform temporal concordance, but in (Vo et al., 2009) plasma testosterone levels were decreased at GD21, while hypospadias was diagnosed in adult rats (PND63), long after exposure was ceased. In (Li et al., 2017), hypospadias and testosterone levels were assessed/measured at the same time point (GD19), however this is known not to be an optimal time point for hypospadias diagnosis in rats as the penis is not finished developing.

Incidence concordance

The study with DBP supports incidence concordance, because the non-hypospadiac rats exposed to DBP also had lower serum testosterone levels compared to control rats at GD19 (Li et al., 2017). This is however only weak evidence, as hypospadias was not evaluated at an optimal time point.

The uncertainties of the two in vivo studies have been discussed. Both were classified as unreliable in the evaluation of methodological reliability. Of the two studies, the study by (Li et al., 2017) is considered most uncertain due to the timepoint of hypospadias assessment. A study with in utero exposure to an 5α-reductase inhibitor disrupted genital tubercle development at GD19, but hypospadias was not observed at PND90, indicating that assessment prior to birth may not be true indications of postnatal outcomes (Iguchi et al., 1991). The deficiencies in (Vo et al., 2009) are less severe but overall poses an uncertainty to the study due to missing key information about study design.

The uncertainties in the human evidence mainly pertains to the fact that testosterone levels were not measured during fetal life but in newborn or juvenile males. Given that most cases involve genetic mutations, which occur early in embryonic life, it is highly likely that the effects of mutations on testosterone levels manifest early in fetal life. Another uncertainty with the human cases which include mutations is that it cannot be excluded that the hypospadias phenotype is caused by the low testosterone levels and not directly by genetic mutation.

Another uncertainty for this KER from the literature is the observation that humans with 5α-reductase deficiency have hypospadias due to low DHT levels despite normal or higher testosterone levels (Mendonca et al., 1996). This indicates that the effects of low testosterone may be more through reduced conversion to DHT than due to a direct loss of testosterone action on AR. To this, there is also the existence of a “backdoor pathway” to DHT in humans. This pathway in peripheral tissues (i.e. not testes) can circumvent testosterone as a precursor for DHT by synthesis of DHT is from reduction of androsterone by 17β-HSD (Miller & Auchus, 2019). This would create the possibility that testosterone is not required for DHT production and ultimately AR activation.

Quantitative Understanding of the Linkage

The quantitative understanding of this KER is low.

The time-scale for this KER depends on species but is likely weeks. Testosterone is secreted from around GW8 in humans (GD16 in rats), marking the beginning of the masculinization programming window and programming of the genital tubercle. Hypospadias is diagnosed at birth in humans (Yu et al., 2019) and can also be observed at birth in rodents, but as development of the penis continues after birth in rodents, hypospadias may be more optimally evaluated later in juvenile or adult male rats (Schlomer et al., 2013; Sinclair et al., 2017).

There are no known modulating factors for this KER.

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

References

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Relationship: 2828: Decrease, AR activation leads to Hypospadias

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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.

Key Event Relationship Description

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 Supporting this KER

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).

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.

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.

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).

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.

Quantitative Understanding of the Linkage

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.

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).

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). 

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

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