Relationship: 2130: Antagonism, Androgen receptor leads to Decrease, AR activation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

This KER is applicable to mammals as AR expression and activity is highly conserved (Davey & Grossmann, 2016). AR activity is important for sexual development and reproduction in both males and females (Prizant et al., 2014; Walters et al., 2010). AR function is required during development, puberty, and adulthood. 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-activated steroid hormone nuclear receptor (Davey & Grossmann, 2016). In its inactive state, the AR locates to the cytoplasm (Roy et al., 2001). When activated, the AR translocates to the nucleus, dimerizes, and, together with co-regulators, binds to specific DNA regulatory sequences to regulate gene transcription (Davey & Grossmann, 2016) (Lamont and Tindall, 2010). This is considered the canonical AR signaling pathway. The AR can also activate non-genomic signalling (Jin et al., 2013). However, this KER focuses on the canonical pathway.

The two main AR ligands are the androgens testosterone (T) and the more potent dihydrotestosterone (DHT). Androgens bind to the AR to mediate downstream androgenic responses, such as male development and masculinization (Rey, 2021; Walters et al., 2010). Antagonism of the AR would decrease AR activation and therefore the downstream AR-mediated effects.  

Evidence Supporting this KER

The biological plausibility for this KER is considered high.

The AR belongs to the steroid hormone nuclear receptor family. The AR has 3 main domains essential for its activity, the N-terminal domain, the ligand binding domain, and the DNA binding domain (Roy et al., 2001). Ligands, such as T and DHT, must bind to the ligand binding domain to activate AR allowing it to fulfill its role as a transcription factor. The binding of the ligand induces a change in AR conformation allowing it to translocate to the nucleus and congregate into a subnuclear compartment (Marcelli et al., 2006; Roy et al., 2001) homodimerize and bind to the DNA target sequences and regulate transcription of target genes. Regulation of AR target genes is greatly facilitated by numerous co-factors. Active AR signaling is essential for male reproduction and sexual development and is also crucial in several other tissues and organs such as ovaries, the immune system, bones, and muscles (Ogino et al., 2011; Prizant et al., 2014; Rey, 2021; William H. Walker, 2021).

AR antagonists can compete with or prevent in different ways  AR ligand binding, thereby preventing AR activation. Antagonism of the AR can prevent translocation to the nucleus, compartmentalization, dimerization and DNA binding. Consequently, AR cannot regulate transcription of target genes and androgen signalling is disrupted. This can be observed using different AR activation assays such as AR dimerization, translocation, DNA binding or transcriptional activity assays (Brown et al., 2023; OECD, 2020).

The empirical evidence for this KER is considered high

The effects of AR antagonism have been shown in many studies in vivo and in vitro.

Several stressors can act as antagonists of the AR and lead to decreased AR activation. Some of these are detailed in an AOP key event relationship report by (Pedersen et al., 2022) and shown below, exhibiting evidence of dose-concordance:

Stressors

• Cyproterone acetate: Using the AR-CALUX reporter assay in antagonism mode, cyproterone acetate showed an IC50 of 7.1 nM (Sonneveld, 2005)
• Epoxiconazole: Using transiently AR-transfected CHO cells, epoxiconazole showed a LOEC of 1.6 µM and an IC50 of 10 µM (Kjærstad et al., 2010).
• Flutamide: Using the AR-CALUX reporter assay in antagonism mode, flutamide showed an IC50 of 1.3 µM (Sonneveld, 2005).
• Flusilazole: Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.8 µM and an IC50 of 2.8 (±0.1) µM (Draskau et al., 2019).
• Prochloraz: Using transiently AR-transfected CHO cells, prochloraz showed a LOEC of 6.3 µM and an IC50 of 13 µM (Kjærstad et al., 2010).
• Propiconazole: Using transiently AR-transfected CHO cells, propiconazole showed a LOEC of 12.5 µM and an IC50 of 18 µM (Kjærstad et al., 2010).
• Tebuconazole: Using transiently AR-transfected CHO cells, tebuconazole showed a LOEC of 3.1 µM and an IC50 of 8.1 µM (Kjærstad et al., 2010).
• Triticonazole: Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.2 µM and an IC50 of 0.3 (±0.01) µM (Draskau et al., 2019).
• Vinclozolin: Using the AR-CALUX reporter assay in antagonism mode, vinclozolin showed an IC50of 1.0 µM(Sonneveld, 2005).”(Pedersen et al., 2022)

Other evidence:

Known AR antagonists are used for treatment of AR-sensitive cancers such as flutamide for prostate cancer (Mahler et al., 1998).

Known antiandrogenic compounds like hydroxyflutamide have been shown to act as agonists when the AR carries certain mutations, therefore contributing to uncertainties (Yeh et al., 1997). Additionally, the levels of endogenous androgens (e.g., testosterone or dihydrotestosterone) and the variability in the presence and function of AR co-activators may modulate the effect of AR antagonism.

Quantitative Understanding of the Linkage

 The quantitative relationship between AR antagonism and AR activation will depend on the type of antagonist.

Nuclear translocation in HeLa cells transfected with AR-GFP show a response within 2 hours after ligand exposure (Marcelli et al., 2006; Szafran et al., 2008). Another assay focusing on AR binding to promoters in LNCaP cells has shown that after ligand binding, AR is able to translocate and bind to the DNA sequences within 15min showing the speed of AR activation (Kang et al., 2002).

AR antagonism can lead to increased AR transcript stability and levels as a compensatory mechanism in prostate cancer cells (Dart et al., 2020). In turn, in presence of increased AR levels, AR antagonists can exhibit agonistic activity (Chen et al., 2003).

References

Brown, E. C., Hallinger, D. R., Simmons, S. O., Puig-Castellví, F., Eilebrecht, E., Arnold, L., & Bioscience, P. A. (2023). High-throughput AR dimerization assay identifies androgen disrupting chemicals and metabolites. Front. Toxicol, 5, 1134783. https://doi.org/10.3389/ftox.2023.1134783

Chen, C. D., Welsbie, D. S., Tran, C., Baek, S. H., Chen, R., Vessella, R., Rosenfeld, M. G., & Sawyers, C. L. (2003). A R T I C L E S Molecular determinants of resistance to antiandrogen therapy. NATURE MEDICINE, 10(1). https://doi.org/10.1038/nm972

Dart, D. A., Ashelford, K., & Jiang, W. G. (2020). AR mRNA stability is increased with AR-antagonist resistance via 3′UTR variants. https://doi.org/10.1530/EC-19-0340

Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. In Androgen Receptor Biology Clin Biochem Rev (Vol. 37, Issue 1).

Draskau, M. K., Boberg, J., Taxvig, C., Pedersen, M., Frandsen, H. L., Christiansen, S., & Svingen, T. (2019). In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole. Environmental Pollution, 255, 113309. https://doi.org/10.1016/j.envpol.2019.113309

Jin, H. J., Kim, J., & Yu, J. (2013). Androgen receptor genomic regulation. In Translational Andrology and Urology (Vol. 2, Issue 3, pp. 158–177). AME Publishing Company. https://doi.org/10.3978/j.issn.2223-4683.2013.09.01

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

Kjærstad, M. B., Taxvig, C., Nellemann, C., Vinggaard, A. M., & Andersen, H. R. (2010). Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. Reproductive Toxicology, 30(4), 573–582. https://doi.org/10.1016/j.reprotox.2010.07.009

Lamont, K. R., and Tindall, D. J. (2010). Androgen Regulation of Gene Expression. Adv. Cancer Res. 107, 137–162. doi:10.1016/S0065-230X(10)07005-3.

Mahler, C., Verhelst, J., and Denis, L. (1998). Clinical pharmacokinetics of the antiandrogens and their efficacy in prostate cancer. Clin. Pharmacokinet. 34, 405–417. doi:10.2165/00003088-199834050-00005/METRICS.

Marcelli, M., Stenoien, D. L., Szafran, A. T., Simeoni, S., Agoulnik, I. U., Weigel, N. L., Moran, T., Mikic, I., Price, J. H., & Mancini, M. A. (2006). Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility. Journal of Cellular Biochemistry, 98(4), 770–788. https://doi.org/10.1002/jcb.20593

OECD (2020). Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. OECD Guide. Paris: OECD Publishing doi:10.1787/9789264264366-en.

Ogino, Y., Miyagawa, S., Katoh, H., Prins, G. S., Iguchi, T., & Yamada, G. (2011). Essential functions of androgen signaling emerged through the developmental analysis of vertebrate sex characteristics. Evolution & Development, 13(3), 315–325. https://doi.org/10.1111/j.1525-142X.2011.00482.x

Pedersen, E. B., Christiansen, S., & Svingen, T. (2022). AOP key event relationship report: Linking androgen receptor antagonism with nipple retention. Current Research in Toxicology, 3, 100085. https://doi.org/10.1016/j.crtox.2022.100085

Prizant, H., Gleicher, N., & Sen, A. (2014). Androgen actions in the ovary: balance is key. Journal of Endocrinology, 222(3), R141–R151. https://doi.org/10.1530/JOE-14-0296

Rey, R. A. (2021). The Role of Androgen Signaling in Male Sexual Development at Puberty. Endocrinology, 162(2). https://doi.org/10.1210/endocr/bqaa215

Roy, A. K., Tyagi, R. K., Song, C. S., Lavrovsky, Y., Ahn, S. C., Oh, T. S., & Chatterjee, B. (2001). Androgen receptor: Structural domains and functional dynamics after ligand-receptor interaction. Annals of the New York Academy of Sciences, 949, 44–57. https://doi.org/10.1111/j.1749-6632.2001.tb04001.x

Sonneveld, E. (2005). Development of Androgen- and Estrogen-Responsive Bioassays, Members of a Panel of Human Cell Line-Based Highly Selective Steroid-Responsive Bioassays. Toxicological Sciences, 83(1), 136–148. https://doi.org/10.1093/toxsci/kfi005

Szafran, A. T., Szwarc, M., Marcelli, M., & Mancini, M. A. (2008). Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects. PLoS ONE, 3(11), e3605. https://doi.org/10.1371/journal.pone.0003605

Walters, K. A., Simanainen, U., & Handelsman, D. J. (2010). Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. In Human Reproduction Update (Vol. 16, Issue 5, pp. 543–558). Hum Reprod Update. https://doi.org/10.1093/humupd/dmq003

William H. Walker. (2021). Androgen Actions in the Testis and the Regulation of Spermatogenesis. In Advances in Experimental Medicine and Biology: Vol. volume 1381 (pp. 175–203).

Yeh, S., Miyamoto, H., & Chang, C. (1997). ARA70 and androgenic activity of hydroxyflutamide Hydroxyflutamide may not always be a pure antiandrogen (Vol. 349).

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.

Kanno, Yuichiro, Nao Saito, Ryota Saito, Tomohiro Kosuge, Ryota Shizu, Tomofumi Yatsu, Takuomi Hosaka, Kiyomitsu Nemoto, Keisuke Kato, and Kouichi Yoshinari. 2022. “Differential DNA-Binding and Cofactor Recruitment Are Possible Determinants of the Synthetic Steroid YK11-Dependent Gene Expression by Androgen Receptor in Breast Cancer MDA-MB 453 Cells.” Experimental Cell Research 419(2). doi: 10.1016/j.yexcr.2022.113333.

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.

Knapczyk-Stwora, Katarzyna, Anna Nynca, Renata E. Ciereszko, Lukasz Paukszto, Jan P. Jastrzebski, Elzbieta Czaja, Patrycja Witek, Marek Koziorowski, and Maria Slomczynska. 2019. “Flutamide-Induced Alterations in Transcriptional Profiling of Neonatal Porcine Ovaries.” Journal of Animal Science and Biotechnology 10(1):1–15. doi: 10.1186/s40104-019-0340-y.

Lamont, K. R., and Tindall, D. J. (2010). Androgen Regulation of Gene Expression. Adv. Cancer Res. 107, 137–162. doi:10.1016/S0065-230X(10)07005-3.

MacLean, Helen E., W. S. Maria Chiu, Amanda J. Notini, Anna-Maree Axell, Rachel A. Davey, Julie F. McManus, Cathy Ma, David R. Plant, Gordon S. Lynch, and Jeffrey D. Zajac. 2008. “ Impaired Skeletal Muscle Development and Function in Male, but Not Female, Genomic Androgen Receptor Knockout Mice .” The FASEB Journal 22(8):2676–89. doi: 10.1096/fj.08-105726.

Maiuri, Paolo, Anna Knezevich, Alex De Marco, Davide Mazza, Anna Kula, Jim G. McNally, and Alessandro Marcello. 2011. “Fast Transcription Rates of RNA Polymerase II in Human Cells.” EMBO Reports 12(12):1280–85. doi: 10.1038/embor.2011.196.

Mora, Gloria R., and Virendra B. Mahesh. 1999. Autoregulation of the Androgen Receptor at the Translational Level: Testosterone Induces Accumulation of Androgen Receptor MRNA in the Rat Ventral Prostate Polyribosomes.

Peng, Yajie, Hui Zhu, Bing Han, Yue Xu, Xuemeng Liu, Huaidong Song, and Jie Qiao. 2021. “Identification of Potential Genes in Pathogenesis and Diagnostic Value Analysis of Partial Androgen Insensitivity Syndrome Using Bioinformatics Analysis.” Frontiers in Endocrinology 12. doi: 10.3389/fendo.2021.731107.

Rana, Kesha, Barbara C. Fam, Michele V Clarke, Tammy P. S. Pang, Jeffrey D. Zajac, and Helen E. Maclean. 2011. “Increased Adiposity in DNA Binding-Dependent Androgen Receptor Knockout Male Mice Associated with Decreased Voluntary Activity and Not Insulin Resistance.” Am J Physiol Endocrinol Me-Tab 301:767–78. doi: 10.1152/ajpendo.00584.2010.-In.

Roy, Arun K., Rakesh K. Tyagi, Chung S. Song, Yan Lavrovsky, Soon C. Ahn, Tae Sung Oh, and Bandana Chatterjee. 2001. “Androgen Receptor: Structural Domains and Functional Dynamics after Ligand-Receptor Interaction.” Pp. 44–57 in Annals of the New York Academy of Sciences. Vol. 949. New York Academy of Sciences.

Russell, Patricia K., Michele V. Clarke, Jarrod P. Skinner, Tammy P. S. Pang, Jeffrey D. Zajac, and Rachel A. Davey. 2012. “Identification of Gene Pathways Altered by Deletion of the Androgen Receptor Specifically in Mineralizing Osteoblasts and Osteocytes in Mice.” Journal of Molecular Endocrinology 49(1):1–10. doi: 10.1530/JME-12-0014.

Shiina, Hiroko, Takahiro Matsumoto, Takashi Sato, Katsuhide Igarashi, Junko Miyamoto, Sayuri Takemasa, Matomo Sakari, Ichiro Takada, Takashi Nakamura, Daniel Metzger, Pierre Chambon, Jun Kanno, Hiroyuki Yoshikawa, and Shigeaki Kato. 2006. Premature Ovarian Failure in Androgen Receptor-Deficient Mice. Vol. 103.

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Tut, Thein G., Farid J. Ghadessy, M. A. Trifiro, L. Pinsky, and E. L. Yong. 1997. Long Polyglutamine Tracts in the Androgen Receptor Are Associated with Reduced Trans-Activation, Impaired Sperm Production, and Male Infertility*. Vol. 82.

Wang, Ruey Sheng, Shuyuan Yeh, Lu Min Chen, Hung Yun Lin, Caixia Zhang, Jing Ni, Cheng Chia Wu, P. Anthony Di Sant’Agnese, Karen L. DeMesy-Bentley, Chii Ruey Tzeng, and Chawnshang Chang. 2006. “Androgen Receptor in Sertoli Cell Is Essential for Germ Cell Nursery and Junctional Complex Formation in Mouse Testes.” Endocrinology 147(12):5624–33. doi: 10.1210/en.2006-0138.

Welsh, M., L. Moffat, K. Belling, L. R. de França, T. M. Segatelli, P. T. K. Saunders, R. M. Sharpe, and L. B. Smith. 2012. “Androgen Receptor Signalling in Peritubular Myoid Cells Is Essential for Normal Differentiation and Function of Adult Leydig Cells.” International Journal of Andrology 35(1):25–40. doi: 10.1111/j.1365-2605.2011.01150.x.

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Yu, I. Chen, Hung Yun Lin, Ning Chun Liu, Ruey Shen Wang, Janet D. Sparks, Shuyuan Yeh, and Chawnshang Chang. 2008. “Hyperleptinemia without Obesity in Male Mice Lacking Androgen Receptor in Adipose Tissue.” Endocrinology 149(5):2361–68. doi: 10.1210/en.2007-0516.

Yu, Shengqiang, Chiuan Ren Yeh, Yuanjie Niu, Hong Chiang Chang, Yu Chieh Tsai, Harold L. Moses, Chih Rong Shyr, Chawnshang Chang, and Shuyuan Yeh. 2012. “Altered Prostate Epithelial Development in Mice Lacking the Androgen Receptor in Stromal Fibroblasts.” Prostate 72(4):437–49. doi: 10.1002/pros.21445.

Zhang, Caixia, Shuyuan Yeh, Yen-Ta Chen, Cheng-Chia Wu, Kuang-Hsiang Chuang, Hung-Yun Lin, Ruey-Sheng Wang, Yu-Jia Chang, Chamindrani Mendis-Handagama, Liquan Hu, Henry Lardy, Chawnshang Chang, and † † George. 2006. Oligozoospermia with Normal Fertility in Male Mice Lacking the Androgen Receptor in Testis Peritubular Myoid Cells.

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Relationship: 2820: Decrease, AR activation leads to AGD, decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic

Fetal masculinization including the AGD is regulated by androgens interacting with the AR in all mammals, including humans (Murashima et al., 2015; Thankamony et al., 2016), although, the size of the AGD and difference between the sexes vary between species. A large number of studies exist showing that fetal exposure to anti-androgens causes shortened AGD in male rats and mice (Schwartz et al., 2019, see also Table 2).  Some epidemiological studies find associations between exposure to anti-androgenic compounds and shorter AGD in boys (Thankamony et al., 2016). However, the associations are not very clear and confidence in the data is limited by conflicting results, possibly due to differences in study design and methods for exposure measurements and analyses. Nevertheless, the KER is considered applicable to humans, based on current understanding of the role of AR activation in fetal masculinization.

Life stage

Programming of the AGD occurs during the masculinization programming window in fetal life. This takes place in rats around embryonic days 15.5-19.5 (GD16-20) and likely gestation weeks 8-14 in humans (Welsh et al., 2008). It should be mentioned that though AGD is believed to be relatively stable throughout life, it can be responsive to postnatal changes in androgen levels (Schwartz et al., 2019).

Sex

Data presented in this KER support that disruption of androgen action during fetal life can lead to a short AGD in male offspring. While exposure to chemicals during fetal life can also shorten female AGD, the biological significance and the mechanism driving the effect is unknown (Schwartz et al., 2019).

Key Event Relationship Description

This KER refers to a decrease in androgen receptor (AR) activation during fetal development leading to decreased anogenital distance (AGD) in male offspring.
It should be noted that the upstream Key Event (KE) ‘decrease, androgen receptor activation’ (KE-1614 in AOP Wiki) specifically focuses on decreased activation of the androgen receptor in vivo, while most methods that can be used to measure AR activity are carried out in vitro. Indirect information about this KE may for example be provided from assays showing in vitro AR antagonism, decreased in vitro or in vivo testosterone production/levels or decreased in vitro or in vivo dihydrotestosterone (DHT) production/levels.

Evidence Supporting this KER

The biological plausibility for this KER is judged to be high based on the following:

- Sexual differentiation happens in fetal life. The testes are developed and start to produce testosterone that is converted in other tissues by the enzyme 5-alpha-reductase to the more potent androgen dihydrotestosterone (DHT). Both hormones bind and activate the nuclear receptor and transcription factor AR that in turn drives masculinization of the male fetus (Welsh et al., 2014; Schwartz et. al, 2019).

- Fetal masculinization depends on activation of androgen signaling during a critical time window, the masculinization programming window (MPW), from gestational day (GD) 15.5-18.5 in rats, 14.5-16.5 in mice and presumably gestation weeks (GWs) 8-14 in humans (Welsh et al., 2008; Amato et al., 2022). The onset of AR expression in the tissues of the reproductive tract follows the timing of the MPW (Welsh et al., 2008).

- The fetal masculinization process involves a range of tissues and organs, including the perineum. Perineum length can be measured as the AGD, which is the distance between the anus and the genitalia. The AGD is approximately twice as long in male as in female newborn rodents and humans (Schwartz et al., 2019).

- Male AR knockout mice present shorter AGD than wildtype males, so short that it is indistinguishable from wildtype female littermates (Yeh et al., 2002, Sato et al., 2004).

- In human males, mutations decreasing AR activity also lead to feminization. One example is the androgen insensitivity syndrome (AIS), where mutations in the AR lead to an impaired or abolished response to androgens, and thereby some degree of feminization of XY individuals and even XY sex reversal in individuals with complete AIS (CAIS) (Thankamony et al., 2016; Hughes et al., 2012; Crouch et al., 2011). XY individuals with CAIS present as women with internally placed testes. A study showed that the clitoral to urethral distance in these individuals was similar to a control group of women, but it is not clear whether this measurement can work as a proxy for measuring the AGD (Thankamony et al 2016, Crouch 2011). Unfortunately, it seems the AGD has not at present been measured in CAIS individuals. Another example is human males lacking 5-alpha-reductase, also presenting female-like genitalia (Batista & Mendonca, 2022).
 

- The detailed mechanism by which androgens regulate the AGD is not known but it is hypothesized that the AGD is influenced by the size of the levator-ani and bulbocavernosus (LABC) muscle complex in the perineum. The growth of this complex is stimulated by AR activation, it is sexually dimorphic and larger in males than in females and (Schwartz et al., 2019). AR is required for the development of the LABC complex as demonstrated by AR general and muscle specific knockout mice. AR is expressed in non-myocytic cells in the LABC complex, starting at E15.5 in mice, and knockout of AR in these cells results in defects in the muscle formation  (Ipulan et al., 2016;). Differential gene expression profiles in the perineum of male and female rats as well as in antiandrogen-exposed male rats have been identified providing further mechanistic understanding (Schwartz et al, 2019; Draskau et al, 2022).

Animal in vivo data

The empirical support from studies in animals for this KER is overall judged as high.

It should be noted that the KE decreased androgen receptor activation (KE-1614 in AOP Wiki) specifically focuses on decreased activation of the androgen receptor in vivo, with no methods currently available to measure this. Examples of assays that provide indirect information about KE-1614 are described in upstream MIE/KEs.

The empirical evidence for this KER from animal studies in vivo is based on studies using five different substances that result in decreased AR activation by different mechanisms. Flutamide, procymidone and vinclozolin bind to the AR and inhibit the receptor activity and thereby act as AR antagonists, see MIE26. Finasteride inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT, see MIE1617. DEHP exposure during prenatal development in rats results in reduced fetal testosterone levels, see KE1690. (MIE26, MIE1617 and KE1690 can be found in AOP Wiki).

The evidence for the upstream KE is mainly based on data from in vitro assays (AR antagonism or 5-alpha-reductase inhibition in vitro) whereas the evidence for the downstream KE is based on in vivo studies, and there is generally not evidence for both KEs from the same study. However, decreased testosterone levels can be measured in vivo, and Borch et al., 2004 measured the effect of developmental DEHP exposure on both testosterone levels and AGD (see section about “Dose concordance”).

The empirical animal evidence for the five substances is summarized in table 3.

Table 3. Summary of empirical evidence for decreased androgen receptor activation, leading to decreased male AGD. References for the studies supporting the empirical evidence are found in section “Evidence for decreased AR activation (KE1614) by flutamide, procymidone, and vinclozolin, finasteride and DEHP” and in table 2.

From table 3, it can be deducted that fetal exposure to substances known to decrease androgen receptor activation through antagonism of the AR (vinclozolin, procymidone, flutamide), inhibition of testosterone synthesis (DEHP) or inhibition of conversion of testosterone to DHT (finasteride), results in decreased AGD in rat and mouse male offspring.

Evidence for decreased AR activation (KE 1614) by flutamide, procymidone, vinclozolin, finasteride and DEHP

Flutamide, a pharmaceutical, binds the AR and inhibits the receptor activity, thereby acting as an AR antagonist. It has been used as an antiandrogen for treatment of prostate cancer and is used as a reference chemical for antiandrogenic activity in the AR transactivation assays in the OECD test guideline No 458 (Goldspiel & Kohler, 1990; Labrie, 1993; OECD, 2023; Simard et al., 1986).

Procymidone and vinclozolin are fungicides that have been shown to be AR antagonists. Procymidone binds to the AR and inhibits the agonist binding as shown in AR binding assays using rat prostate cytosol (Hosokawa et al., 1993) or AR transfected COS cells (Ostby et al., 1999). Procymidone also inhibits agonist activated transcription in AR reporter assays (Hass et al., 2012; Kojima et al., 2004; Orton et al., 2011; Ostby et al., 1999; Scholze et al., 2020). Vinclozolin binds to the AR and inhibits the agonist binding as shown in AR binding assays using rat epididymis cytosol (Kelce et al., 1997) or AR transfected COS-1 cells (Wong et al., 1995).
Vinclozolin also inhibits agonist activated transcription in AR reporter assays (Euling et al, 2002; Kojima et al., 2004; Molina-Molina et al., 2006; Orton et al., 2011; Scholze et al., 2020; Shimamura et al., 2002; Wong et al., 1995). Finasteride is a pharmaceutical that inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT. Finasteride is used to treat benign prostatic hypertrophy (Andersson & Russel, 1990; Rittmaster & Wood, 1994; Stoner, 1990).

Prenatal exposure to DEHP in rats results in reduced production of testosterone in fetal testis measured in ex vivo testis assays, reduced testosterone levels in testis and reduced fetal plasma or serum testosterone levels (Borch et al., 2004; Borch et al., 2006; Culty et al., 2008; Hannas et al., 2011; Hannas et al., 2012; Klinefelter et al., 2012; Parks et al., 2000; Wilson et al., 2004; Wilson et al., 2007; Vo et al., 2009). Two studies don’t show an effect on testosterone levels in testis or fetal plasma testosterone levels, respectively (Andrade et al., 2006; Borch et al., 2006). The precise underlying mechanism is presently unknown.

Evidence for decreased AGD in males (KE1688) by prenatal exposure to flutamide, procymidone, vinclozolin, finasteride and DEHP

All datasets that were used for the weight of evidence assessment were judged as reliable without or with restriction. The majority of datasets assessed showed a decreased male AGD. The conclusion was that the level of confidence was strong for all five substances. The studies are summarized in table 4.

Empirical evidence for the included substances

Table 4. Empirical evidence for decreased AGD in males (KE1688) by prenatal exposure to flutamide, procymidone, vinclozolin, finasteride and DEHP. *One dose only.

>>>>>TABLE 4<<<<<

Epidemiological data on DEHP

The biggest relevant epidemiological dataset was identified on associations between DEHP and AGD.  

Six prospective cohort studies and one cross-sectional study on the association between maternal DEHP metabolites and length of AGD (anopenile distance (APD) and anoscrotal distance (ASD)) in boys were assessed as reliable without or with restriction. Decreased AGD (anopenile distance (APD) and/or anoscrotal distance (ASD)) was observed in three prospective cohort studies (Martino-Adrade et al., 2016; Swan et al., 2005 reviewed and updated in Swan 2008; Wenzel et al., 2018). In contrast, no significant association was observed in three other prospective cohort studies (Arbuckle et al., 2018; Henriksen et al., 2023; Jensen et al., 2016) and the cross-sectional study (Sunman et al., 2019). This inconsistency introduces a level of uncertainty regarding the overall association. Therefore, the level of confidence was judged as weak. 

Dose concordance

Dose concordance is challenging to assess for this KER since in vivo AR activity is currently not possible to measure, but only can be informed indirectly by measures of upstream events.

However, some studies provide useful information that support dose concordance between the KEs.

In a publication by Borch et al., rats were exposed in utero to DEHP at GD7-21. Fetal testosterone levels in testes and serum and testosterone production in fetal testes ex vivo were investigated at GD21, whereas AGD was investigated at PND3. The LOAELs for reduced testosterone production in ex vivo fetal testes and reduced testosterone levels in fetal testes were 300 mg/kg/d, whereas the LOAEL for decreased AGD in male offspring was 750 mg/kg/d (Borch et al., 2004). 

In a publication by Scholze et al, AR antagonism and decreased testosterone synthesis was quantitatively assessed (IC50) in vitro for a list of substances. In addition, internal concentrations in male fetuses and effects on AGD were measured after fetal exposure to the same substances. In utero exposure to all the substances lead to reduced AGDIndex (AGDI) in the exposed male offspring. Further, for all substances except Cyprodinil, the internal exposure levels in the fetuses leading to reduced AGD exceeded the IC50 levels observed in one or both of the in vitro assays.
Three different doses of linuron exposure were included. The medium exposure dose led to a higher level of internal exposure and a higher degree of AGDI reduction than the low dose. AGDI could not be determined in the highest dose due to maternal toxicity (Scholze et al., 2020).

Temporal concordance

Temporal concordance can only be considered from a theoretical perspective since the downstream event, decreased AGD, is usually measured at GD21, PND0 or PND1 in rats, and due to the size of the fetuses is not feasible to measure at earlier timepoints.

Considering the biology, the upstream event – decreased AR activation in vivo – is foreseen to happen minutes to hours after exposure. If a substance decreases AR activation through inhibition of the AR, the upstream event is expected to happen immediately after exposure. If a substance decreases androgen receptor activation through inhibition of testosterone synthesis, the upstream event is expected to happen minutes to hours after the exposure, though it is uncertain exactly when the change will be big enough to be measurable. On the other hand, the downstream event – decreased AGD - is a measurement of relative growth of the perineal tissue, which is expected to take days in the developing fetus.

For the model substances, there were some inconsistencies in the empirical evidence, but they could be explained by differences in study designs and uncertainties in measurements, see appendix 1: 8dh20j155i_FINAL_Appendix_KER2820_For_Wiki.pdf.

Species differences in effects of phthalates (including DEHP and DBP) on fetal testes testosterone production have been observed between humans, mice and rats. In human fetal testes exposed to DEHP or DBP in vitro or ex vivo, no suppression of testosterone production is observed, which contrasts observations in rat fetal testes under similar conditions. Also in mice, testosterone production in the fetal testes is unaffected by treatment with DEHP or DBP in vitro or in utero (Sharpe, 2020).

The species differences described above are specific for some phthalates and their interference with fetal testicular testosterone production. This uncertainty should not be reflected on other antiandrogenic substances, especially not those acting through other mechanisms of action.
The association between exposure to DEHP and reduced AGD in humans is judged to be weak, which may further support a species difference between rodents and humans, but it may also reflect the large uncertainties inherent in the epidemiological studies.

Observational epidemiological studies face challenges in proving cause-effect relationships as they cannot control conditions like experimental animal and in vitro studies. Human studies can identify associations between variables but cannot offer conclusive proof of causation (Lanzoni et al., 2019). Various study designs and statistical methods are employed to strengthen evidence within the inherent limitations of observational research (Song & Chung, 2010; Olier et al., 2023). Inconsistencies in epidemiological data arise from various factors, such as different methodologies used in exposure and outcome measurement and also in statistical analyses.

These differences collectively contribute to the complexity of interpreting and weighing the evidence in epidemiological research.

Quantitative Understanding of the Linkage

The quantitative understanding of the linkage is low. This is a consequence of it not being possible to measure the upstream and the downstream event in the same study.

In one study, a quantitative model was developed to predict the decrease in AGD from in vitro AR antagonism or in vitro decreased testosterone synthesis. The authors conclude that predicting the effect on AGD in vivo based on the in vitro results is only possible on a qualitative level, but the model cannot predict AGD reductions quantitatively (Scholze et al., 2020).

AR activation operates on a time-scale of minutes. The AR is a ligand-activated nuclear receptor and transcription factor. Upon ligand binding a conformational change and subsequent dimerization of the AR takes place within 3-6 minutes (Schaufele et al., 2005). Nuclear translocation (Nightingale et al., 2003) 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).

For the downstream event, the time-scale for observing a measurable effect on growth of a tissue (in this case the perineum) is closer to days and weeks depending on species. For instance, in humans, the masculinization programming window is presumed to start around GW 8, while a sexual dimorphism of the AGD can first be observed from around GWs 11-13 (Thankamony et al., 2016) and reaches its maximum 2-fold difference around GWs 17-20 (Sharpe, 2020). 

It has been demonstrated that exposure to flutamide for one day (Foster & Harris, 2005) or vinclozolin for two days (Wolf et al., 2000) during the sensitive window of exposure can elicit a detectable decrease in the AGD in male rat offspring.

A well established modulating factor is genetic variations in the AR which decrease the function of the receptor. For example, longer CAG repeat lengths have been associated with decreased AR activation (Tut et al 1997, Chamberlain et al 1994) and a shorter AGD in adult men (Eisenberg et al., 2013). Other modulating factors being discussed in the literature is maternal age and parity (Barrett et al., 2014), but these associations are only suggestive with more studies needed to confirm the associations (Barrett et al., 2014).

Not relevant for this KER.

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Relationship: 2127: Altered, Transcription of genes by the AR leads to AGD, decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Species

This KER applies to humans, mice, and rats based on biological plausibility. Current empirical evidence is from rat studies only.

Fetal masculinization including the AGD is regulated by androgens interacting with the AR in all mammals, including humans (Murashima et al., 2015; Thankamony et al., 2016), although, the size of the AGD and difference between the sexes vary between species. A large number of studies exist showing that fetal exposure to anti-androgens causes shortened AGD in male rats and mice (Schwartz et al., 2019a).  Some epidemiological studies find associations between exposure to anti-androgenic compounds and shorter AGD in boys (Thankamony et al., 2016). However, the associations are not very clear and confidence in the data is limited by conflicting results, possibly due to differences in study design and methods for exposure measurements and analyses. Nevertheless, the KER is considered applicable to humans, based on current understanding of the role of AR activation in fetal masculinization.

Life stage

The length of the AGD is programmed during fetal life during the masculinization programming window. This takes place in rats around embryonic days 15.5-19.5 (GD16-20) and likely gestation weeks 8-14 in humans (Welsh et al., 2008). It should be mentioned that though AGD is believed to be relatively stable throughout life, it can be responsive to postnatal changes in androgen levels (Schwartz et al., 2019a).

Sex

A decrease in the male AGD is a consequence of disrupted androgen action (Welsh et al 2008). While exposure to chemicals during fetal life can also shorten female AGD, the biological significance and the mechanism driving the effect is unknown (Schwartz et al 2019a).

Key Event Relationship Description

During male reproductive development, the androgen receptor (AR) regulates gene transcription in target tissues to induce masculinization. Target tissues include the perineum, the tissue located between the anus and the genitals. This tissue is sexually dimorphic, with males developing the levator ani-bulbocavernosus (LABC) muscle complex in response to androgen signaling. The anogenital distance (AGD) is about twice as long in newborn males than in females in many mammals such mice, rats and humans.

A consequence of reduced androgen action during the masculinization programming window in utero, the male AGD will end up being shorter, approaching female AGD when AR signaling is almost blocked. Measuring of the AGD thus serves as a morphometric biomarker for compromised androgen action during fetal life and is used in OECD test guidelines for assessing endocrine disruption.

This KER refers to a tissue-specific, in this case the perineum, alteration in AR-mediated gene transcription during fetal development leading to a decreased AGD in male offspring.

Evidence Supporting this KER

Sexual differentiation initiates during fetal life when a surge in testosterone induces masculinization of a range of tissues and organs (Welsh et al). Testosterone and the more potent metabolite DHT mediate masculinization via activation of the AR; a nuclear transcription factor. Androgens thus induce masculinization via altered AR gene transcription in target tissues. This includes the perineum (Niel et al 2008; Ipulan et al 2014) which can be measured as the AGD and is approximately twice as long in newborn male rodents and humans compared to female (Schwartz et al 2019a). This is also evident in male AR knockout mice which present with an AGD that is indistinguishable from wildtype female littermates (MacLean et al 2008; Notini et al 2005).

Current evidence for direct transcriptional changes mediated by AR disruption in the perineum leading to shorter male AGD is limited. Two studies were identified investigating the transcriptional footprint in the perineum after anti-androgen exposure:

Gestational exposure of rats to the 5α-reductase inhibitor finasteride (leading to decreased DHT levels) decreased fetal male AGD with 37% at gestational day (GD) 21. Microarray was used to compare transcriptional profiles between control males, finasteride-exposed males, and control females, revealing a sexually dimorphic transcriptional profile of the perineum, with the profile of finasteride-exposed males being intermediary to the male and female control groups (Schwartz et al 2019b).

Gestational exposure of rats to the AR antagonist triticonazole induced decreased fetal male AGD at GD21 and a differentially expressed set of genes investigated by whole transcriptome sequencing in the perineum at both GD17 and GD21 (Draskau et al 2022).  

References

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Ipulan LA, Suzuki K, Sakamoto Y, Murashima A, Imai Y, Omori A, Nakagata N, Nishinakamura R, Valasek P and Yamada G (2014). Nonmyocytic androgen receptor regulates the sexually dimorphic development of the embryonic bulbocavernosus muscle. Endocrinology 155(7):2467-79. doi: 10.1210/en.2014-1008

MacLean HE, Chiu WS, Notini AJ, Axell AM, Davey RA, McManus JF, Ma C, Plant DR, Lynch GS and Zajac JD (2008). Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice. FASEB J 22(8):2676-89. doi: 10.1096/fj.08-105726

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Niel L, Willemsen KR, Volante SN and Monks DA (2008). Sexual dimorphism and androgen regulation of satellite cell population in differentiating rat levator ani muscle. Dev Neurobiol 68(1):115-22. doi: 10.1002/dneu.20580

Notini AJ, Davey RA, McManus JF, Bate KL and Zajac JD (2005). Genomic actions of the androgen receptor are required for normal male sexual differentiation in a mouse model. J Mol Endocrinol 35(3):547-55. doi: 10.1677/jme.1.0188

Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U and Svingen T (2019a). Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. Arch Toxicol 93(2):253-272. doi: 10.1007/s00204-018-2350-5

Schwartz CL, Vinggaard AM, Christiansen S, Darde TA, Chalmel F and Svingen T (2019b). Distinct Transcriptional Profiles of the Female, Male, and Finasteride-Induced Feminized Male Anogenital Region in Rat Fetuses. Toxicol Sci 169(1):303-311. doi: 10.1093/toxsci/kfz046

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Welsh M, Saunders PT, Fisken M, Scott HM, Hutchison GR, Smith LB, Sharpe RM. Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J Clin Invest 118(4):1479-90. doi: 10.1172/JCI34241