Anogenital distance as a biomarker for incomplete masculinization: Molecular mechanisms in the perineum

Camilla Lindgren Bech*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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Abstract

In both humans and rodents, development of the male reproductive organs and general masculinization of the fetus depends on androgen signaling. This signaling needs to take place within a short time window of fetal life, referred to as the masculinization programming window (MPW). In many respects, androgen action in this time window lays the foundation for male reproductive health later in life. Fetal androgen action also stimulates the growth of the perineal muscles and thereby influence the length of the perineum, i.e. the anogenital distance (AGD). This distance, from the anus to the external genitalia, is directly related to fetal androgen action and is twice as long in males as in females. Consequently, a short male AGD at birth is considered a unique biomarker of disrupted fetal androgen action in both rodents and humans. A short AGD is associated with adverse effects on male reproductive health such as cryptorchidism and hypospadias at birth as well as poor sperm quality and reduced fertility in adulthood. This morphometric measure is therefore used in a regulatory context to test chemicals for their endocrine disruptive properties.
Although we know that androgen signaling is critical for perineal growth, our mechanistic knowledge remains limited. In addition, some chemicals affect AGD in ways that cannot be predicted based on their anti-androgenic potential in vitro. This suggests that other regulatory pathways also play a role in determining the AGD outcome, at least as modifiers of the androgen-mediated effect. Knowledge of the regulatory pathways and molecular events that control perineal development is essential when extrapolating from rodent toxicity studies to humans. In addition, such knowledge could contribute to future work on the development of adverse outcome pathways (AOPs) and new, refined non-animal test strategies in chemical risk assessment. The aim of this PhD project was therefore to identify potential additional signaling pathways affected in the perineal tissues of males with short AGD.
An extensive literature search was conducted to collate the available literature pertaining to the relationship between xenobiotic exposure and effects on the AGD. For the experimental work, two different approaches were used. First, in utero exposure to known anti-androgenic compounds was used to induce short AGD in the male offspring and compare the transcriptional profile of the perineum to that of the control males and the control females. Second, perineum from male and female offspring at different developmental stages was used to investigate the mechanisms that govern the normal, sex-specific development of the perineum.
In the first study, in utero exposure to the 5α-reductase inhibitor finasteride was used to induce a short AGD in the male fetuses at gestational day 21. Microarray analysis of the perineum revealed that the control males, control females and exposed males each had their distinct transcriptional profile. Importantly, the transcriptional profile of the perineum from the exposed males was more closely related to the control females than the control males. This confirms that the short, feminized male AGD is associated with a feminized transcriptional profile of the perineal tissues. Four genes related to Wnt and estrogen signaling (Wnt2, Sfrp4, Esr1, and Padi2) had a sexually dimorphic expression pattern and expression of the estrogen related factors (Esr1 and Padi2) were affected in the finasteride exposed males. These pathways could therefore play a role in the sex-specific development of the perineal tissues. The identification of the four Wnt and estrogen related factors provided targets for further studies on the molecular events governing perineal development.
The second study investigated if the Wnt and estrogen related factors identified in the first study were affected by exposure to other anti-androgenic chemicals. The expression of these factors in the perineum was analyzed following in utero exposure to enzalutamide, vinclozolin and procymidone. Out of these three compounds, the androgen receptor antagonist enzalutamide had the most marked effect on fetal AGD and also had significant effect on perineal expression of Sfrp4. For vinclozolin and procymidone, two pesticides with known anti-androgenic properties, smaller effects were seen on AGD at the doses used but no statistically significant effects on perineal expression Wnt2, Sfrp4, Esr1, and Padi2. These results, combined with the knowledge from the first study with finasteride, suggest that substantial effects on AGD are necessary to detect effects at the transcript level in the perineal tissues. However, all the tested anti-androgenic compounds showed indications of a similar mode of action on the perineum, although this could not be proven by statistical methods due to a low power and/or large biological variation. Together with the available literature, the results from the first two studies could suggest that Wnt and estrogen signaling may modulate AGD outcome by regulating cell proliferation and differentiation in the fetal perineal tissues.
The last study focused on the normal, sexually dimorphic development of the perineum. Here, gene expression was investigated in perineal tissues from male and female fetuses at different stages of development. The results suggest that there is a male-specific increase in non-myocytic progenitor cells in the perineum during early development. These cells provide the basis for differentiation into muscle cells and thus a male-specific increase in myogenic cells at later stages of fetal development. These are very preliminary data and should be followed up by larger, more detailed studies using techniques suited for tracking cell populations in a spatiotemporal context.
Collectively, the studies of this PhD project show that the sexually dimorphic AGD is associated with a sexually dimorphic transcriptional profile of the perineum. Furthermore, anti-androgenic chemicals that induce short male AGD induce a concomitant feminization of the perineal transcriptional profile. The Wnt and estrogen signaling pathways provide targets for future research in perineal development as they are expressed in a sexually dimorphic pattern and are affected in the perineum of males with short AGD following exposure to potent anti-androgens. In addition, the present findings suggest that the sex-specific size difference of the perineal muscles may be based on differences in progenitor cell populations and their subsequent differentiation into muscle cells.
This PhD thesis identifies new possible signaling pathways that may contribute to the development of the perineum and AGD. It highlights possible targets and new approaches to be used in future investigations that may ultimately contribute to describing the detailed adverse outcome pathway (AOP) for chemically induced effects on AGD.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages171
Publication statusPublished - 2020

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