Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring

Monica K. Draskau; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Marie L. Holmer; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Emilie Elmelund; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Anna O. Bindel; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

Terje Svingen; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark

This AOP links decreased intratesticular testosterone levels with short anogenital distance (AGD) in male offspring. It does not yet contain an MIE, as several upstream mechanisms can lead to ‘reduced testosterone’ synthesis in fetal testis, such as inhibiting key steroidogenic enzymes. Testosterone is synthesized from cholesterol through several enzymatic steps, including those catalyzed by CYP enzymes such as CYP11 and CYP17. Once synthesized, testosterone is released into circulation and transported to target tissues where it initiates masculinization by binding to and activating the androgen receptor (AR) in target cells. Notably, testosterone can be converted to DHT by 5α-reductase, with DHT being a more potent AR agonist than testosterone; this testosterone-to-DHT conversion is critical during development for differentiation of male traits, including masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex (Davey and Grossmann, 2016; Keller et al, 1996; Robitaille and Langlois, 2020). The LABC complex fails to develop in the absence or insufficiency of androgen signaling, as for instance observed in female fetuses.

A short AGD around birth is a marker for undervirilization of male fetuses and is associated with male reproductive disorders, including reduced fertility in adulthood (Schwartz et al, 2019). Although a short AGD is not necessarily ‘adverse’ from a human health perspective, it is considered an ‘adverse outcome’ in OECD test guidelines; AGD measurements are mandatory in specific tests for developmental and reproductive toxicity in chemical risk assessment (TG 443, TG 421/422, TG 414), with measurement guidance provided in OECD guidance documents 43 (OECD, 2008) and 151 (OECD, 2013).

A central event in this pathway is the inhibition of testosterone synthesis in the fetal testes, leading to reduced circulating testosterone levels and decreased DHT convertion by 5α-reductase. Insufficient DHT fails to effectively activate AR in target tissues, including the developing perineal region, which leads to failure to properly masculinize the perineum/LABC complex and ultimately a short AGD.

Androgen signaling is critical for male sex differentiation during fetal life, and suboptimal signaling during critical life stages leads to under-masculinized offspring. Androgens, primarily testosterone and DHT, exert their effects by binding to and activating the AR is target cells. Blocking the AR basically blocks androgen signaling and masculinization of tissues that otherwise should masculinize in male fetuses.  One morphometric marker for reduced fetal androgen action is shorter AGD compared to control males.

For the AOP network development, the OECD AOP Developer’s Handbook was followed alongside pragmatic approaches (Svingen et al., 2021). Key events (KEs) and key event relationships (KERs) based on canonical knowledge from the ‘upstream anti-androgenic network’ (Draskau et al., 2024) were treated less stringently, while new units adhered to more rigorous systematic literature retrieval methods. An exception was the new canonical KER-3348 linking KE-3398 (decreased intratesticular testosterone) with KE-1690 (decreased circulating testosterone), which was developed using reviews and selected primary articles as examples.

KER-2820, linking KE-1614 (decreased AR activation) with AO-1688 (decreased AGD), was developed using a systematic weight of evidence (WoE) approach (Holmer et al., 2024). From an initial 826 publications, 557 were retained, with 71 selected for data extraction (82 datasets). Ultimately, 25 reliable datasets from in vivo studies on five model compounds (flutamide, procymidone, vinclozolin, finasteride, di-2-ethylhexyl phthalate) provided strong empirical support.

KER-3449, linking KE 2298 (decreased intratesticular testosterone) with AO-1688 (decreased AGD), and KER-3349, linking KE-1690 (decreased circulating testosterone) with AO-1688 (decreased AGD) were developed systematically (WoE approach) in parallel using the same search string to retrieve all articles pertaining to reduced testosterone and AGD. From an initial 649 publications, 40 in vivo studies were selected for data extraction and reliability evaluation. Of these, 24 reliable data sets were retrieved for KER-3449, and 10 reliable data sets were retrieved for KER-3349, providing strong empirical support for both KERs.

Conversely, KER-2127, linking KE-286 (altered AR transcription) with AO-1688, was developed semi-systematically, yielding only two relevant studies due to limited transcriptional data from perineal tissue exposed to anti-androgenic chemicals.

The overall AOP assessments followed the AOP Developer’s Handbook guidelines.

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