**4. Androgen signaling and placenta function**

To exert a cellular response, steroid hormones need to bind to either a membrane receptor or an intracellular, nuclear or cytoplasmic receptor. T and DHT can bind to either type of receptor, AR (encoded by *AR*, in the human Xq11-12), or a membranebound receptor, such as G protein-coupled receptor family C group 6 member A (GPRC6A) [19]. DHEA and A4 require conversion to T or DHT to exert their androgenic effects.

AR is expressed at all levels of the female hypothalamic-pituitary-gonadal axis, including the brain, ovarian stroma, ovarian follicles and corpus luteum. Furthermore, AR is present in first trimester and term placenta, and localizes to the cytosol of placental villi, and in cytotrophoblast, differentiated syncytiotrophoblast, and placental stroma [20]. In ruminant placentomes, nuclear signals are predominantly observed in invasive TGC and uninucleate trophoblast cells, stromal cells of the chorionic villi, caruncular epithelial, and stromal cells during late gestation [21, 22].

AR belongs to the steroid hormone intracellular receptor family. Exon 1 of the *AR* gene encodes the N-terminal domain, which contains an activation function 1 (AF1) region that interacts with coregulatory proteins to enhance transcriptional regulation of AR target genes. Exons 2 and 3 encode two distinct zinc-fingers (DNA-binding domain) required for interaction with a palindromic androgen response elements (ARE) of the core sequence, 5′-TGTTCT-3′, separated by 3 nucleotides located within the promoter regions of AR target genes. The remaining exons encode a hinge region which contains the nuclear localization signal, and the ligand binding domain [23].

When localized to the cytoplasm, AR is bound by a number of chaperone proteins including heat shock protein 90 (Hsp90) as well as immunophilins. When ligands, T or DHT, bind to AR, there is a conformational change which exposes the nuclear localization signal, allowing the interaction with importin-α, which facilitates nuclear translocation. Once inside the nucleus, two subunits of the AR dimerize and bind the ARE on promoter regions of AR-target genes, resulting in transcriptional regulation, leading to either activation or suppression of expression. Co-regulatory proteins, such as histone lysine demethylases (KDMs), modulate transcriptional activity of AR-target genes. In sheep for example, KDMs have been found to act as co-regulators in trophoblast cells [22, 24]. This interaction with regulator factors is critical for signaling processes in the placenta.

Androgens are known to stimulate proliferation of human umbilical vein endothelial cells, indicating a key role for androgens during pregnancy. During establishment of pregnancy, androgens play a role in embryo implantation. Early in pregnancy, before implantation, T is converted to DHT which regulates transcription of factors necessary for initiation of decidualization and early endometrial receptivity. Near the time of implantation, T itself promotes endometrial remodeling, and soon after implantation it serves as an important precursor for E2 which regulates vascular remodeling [25]. Studies in mice reveal that insufficient androgens may delay embryo implantation, whereas excess androgens lead to aberrant gene expression at implantation sites.

Studies on ovine placentas revealed vascular endothelial growth factor A (VEGFA) expression to be androgen responsive, and androgens are thought to regulate the expression of VEGFA and play a key role in placental angiogenesis [21, 23]. More specifically, AR and the KDM1A coregulator are recruited to an ARE in the ovine VEGFA promoter. On gestational day 90, placenta VEGFA mRNA and VEGFA and AR protein levels increased in testosterone-treated ewes compared to control placentas [22].

**Figure 3.** *Androgen signaling through GPRC6A in target tissue. Image created with BioRender.com.*

In addition to the classical genomic intracellular AR mediated signaling pathways, androgens also act through membrane receptors. GPRC6A is a G protein-coupled receptor (GPCR) that functions as a membrane receptor for small amino acids, cations, osteocalcin, and androgens [26]. GPRC6A is known to have a long extracellular domain to allow for the binding of these different ligands [26]. GPRC6A mediates the effects of osteocalcin, a protein hormone released by osteoblasts, and results in the activation of the cAMP pathway and subsequently, testosterone synthesis by the Leydig cells of the testis. GPRC6A ligand binding can result in the activation of the Gαs, Gαi, and Gαq pathways (**Figure 3**). The presence of GPRC6A has been identified in placental trophoblast cell membranes, indicating the possibility of androgens eliciting a non-genomic effect on cells of the placenta.

Another membrane receptor that androgens elicit a non-genomic effect through is Zrt- and Irt-like protein 9 (ZIP9) [27]. ZIP9 is a zinc transporter that also acts as a receptor for androgens via G-protein coupling. Studies have revealed the presence of ZIP9 in ovarian tissue of Atlantic croakers, and act as a receptor for androgens inducing apoptosis in follicular cells, as well as promoting zinc uptake. Studies also show a similar action in breast and prostate tissue [28]. Ultimately, these two studies reveal that androgens binding to ZIP9 results in the activation of pro-apoptotic genes and the regulation of zinc homeostasis within target tissues [28].

Similarly, Transient receptor potential cation channel subfamily M (melastatin) member 8 (TRPM8) and Oxoeicosanoid receptor 1 (OXER1) bind a variety of ligands including androgens [29]. However, their expression during pregnancy or in placental cells is currently unknown.

### **5. Androgens and pregnancy**

Androgens play a fundamental role in female physiology, particularly during pregnancy. In women, androgens are synthesized by cells within the ovaries, the adrenal glands, fat, and also in placenta, acting in an endocrine or paracrine fashion [30]. DHEA, mainly from the adrenal glands, acts as a crucial precursor for E2 and T in the ovary and other target tissues such as fat [31]. Depending on the intracellular availability of steroidogenic enzymes in target tissues, DHEA is converted to A4

which is a precursor for T, both of which can be aromatized to estrogens [32]. Some studies have reported an elevated level of T during pregnancy. An increase in T levels occurs from the first trimester of pregnancy, becoming more pronounced towards the third trimester, being three-folds higher than observed in non-pregnant women (**Table 1**) [36]. In contrast, maternal circulating DHEA levels decrease in pregnant women due to it being converted to T and E2 [37].

Steroid production varies widely among species, with these differences becoming more pronounced during pregnancy. Each species have their own distinct pattern of steroid serum levels, steroidogenic enzymes, receptors, and transporters to support their individual physiological requirements. For example, in dairy cows, maternal serum T levels increase ~100-fold during the last trimester of gestation (**Table 1**), as well as a ~50-fold increase in milk testosterone levels [38].

In the horse, T elevation during pregnancy presents a biphasic curve (**Table 1**). The first elevation is caused by luteal androgen production, which is stimulated by equine chorionic gonadotropin (eCG). The late rise and fall are temporally related to the development and regression, respectively, of the fetal gonads. The equine placenta has little capacity to synthesize androgens, as it lacks CYP17A1. Hence, androgens in the form of DHEA are substrates for E2 synthesis, and must be supplied mostly by fetal gonads, forming a true feto-placental unit [39].

As androgen levels increase during pregnancy, the mother and developing fetuses usually are protected from excess bioactive androgens by increased secretion of sex hormone-binding and placental aromatase, which converts T into E2. Hyperandrogenism can result from a number of conditions, the most common being luteomas and theca-lutein cysts within the ovary. Luteomas are benign tumors that occur during pregnancy with excess androgen production in 25-35% of the cases [33, 40, 41]. These often go unnoticed and in most cases are non-virilizing.


#### **Table 1.**

*Testosterone serum levels during pregnancy in human [33], horses [34], and cows [35].*

#### *Androgen Signaling in the Placenta DOI: http://dx.doi.org/10.5772/intechopen.94007*

Additional causes of excess androgen production during pregnancy are conditions such as polycystic ovarian syndrome (PCOS), one of the most common endocrine disorders in women of reproductive age, and congenital adrenal hyperplasia (CAH). Both conditions result in pregnancy complications, including pregnancy induced hypertension and pre-eclampsia, a human pregnancy syndrome characterized by the onset of hypertension and proteinuria after 20 weeks of gestation, and can lead to maternal or fetal mortality [42]. In humans, clinical observations have established that women with PCOS exhibit similar features as seen in classical 21-hydroxylase deficiency in CAH, such as anovulation, ovarian hyperandrogenism, LH hypersecretion, polycystic appearing ovaries, and insulin resistance, despite normalization of adrenal androgen excess after birth [43]. Furthermore, animal studies have demonstrated that intrauterine exposure to excessive amounts of androgens can lead to development of PCOS after birth (reviewed in [44, 45]). In fact, prenatal androgenization in pregnant ewes has revealed reproductive and metabolic phenotypes in female offspring that closely resemble PCOS in women. These observations suggest that androgen excess during early life, whether derived from fetal or maternal sources, may provide one possible mechanism to explain the occurrence of PCOS in adulthood.

Less common causes leading to androgen excess during pregnancy include placental aromatase deficiency. Aromatase is encoded by the CYP19A1 gene, and is responsible for converting T to E2. At least 10 different promoters have been identified in its regulatory region, enabling regulation in a tissue-specific manner [46]. Mutations in CYP19A1 prevent aromatization of testosterone, leading to hyperandrogenism and phenotypes similar to androgen excess, including maternal and fetal virilization and development of ambiguous genitalia at birth [47]. Of particular interest is the observation that placental aromatase deficiency is associated with pre-eclampsia [48, 49]. Women with pre-eclampsia have significantly lower levels of placental aromatase, and significantly lower levels of both 17β-estradiol:testosterone and estrone:androstenedione ratios, as well as higher levels of T. In fact, this placental defect in steroidogenesis appears before clinical symptoms of pre-eclampsia and thus may serve as a diagnostic marker.

## **6. Conclusions**

The focus of this chapter was on androgens and their potential role in pregnancy and placental development and function. Normal pregnancy in women is associated with increased maternal serum levels of androgens, which are derived from the adrenal glands, adipose tissue, ovaries, and placenta. Species differences in androgen production exist reflecting species-specific needs for pregnancy maintenance and/or placental function. Furthermore, the placenta contains classical androgen receptors as well as non-classical membrane receptors, indicating the placenta is a target of androgen signaling. Preliminary and ongoing studies suggest a role for androgen signaling in trophoblast cell differentiation and placental angiogenesis.
