Androgens and endometrium: New insights and new targets.

Accepted Manuscript Androgens and endometrium: New insights and new targets Ioannis Simitsidellis, Philippa T.K. Saunders, Douglas A. Gibson PII:

S0303-7207(17)30507-5

DOI:

10.1016/j.mce.2017.09.022

Reference:

MCE 10082

To appear in:

Molecular and Cellular Endocrinology

Received Date: 7 June 2017 Revised Date:

8 September 2017

Accepted Date: 14 September 2017

Please cite this article as: Simitsidellis, I., Saunders, P.T.K., Gibson, D.A., Androgens and endometrium: New insights and new targets, Molecular and Cellular Endocrinology (2017), doi: 10.1016/ j.mce.2017.09.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Androgens and endometrium.

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Androgens and endometrium: new insights and new targets

Ioannis Simitsidellis, Philippa TK Saunders, Douglas A Gibson

Medical Research Council Centre for Inflammation Research, The University of Edinburgh,

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Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK

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Keywords: endometrium, androgen receptor (AR), decidualisation, proliferation, testosterone (T), dihydrotestosterone (DHT), AKR1C3, SRD5A1, scarless healing, endometriosis, cancer.

Word count: 7192

Correspondence: Dr DA Gibson MRC Centre for Inflammation Research, The University of Edinburgh,

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Queen’s Medical Research Institute, 47 Little France Crescent,

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Edinburgh, EH16 4TJ. UK.

Email: [email protected]

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Abstract 25

Androgens are synthesised in both the ovary and adrenals in women and play an important role in the regulation of female fertility, as well as in the aetiology of disorders such as polycystic ovarian syndrome, endometriosis and endometrial cancer. The endometrium is an androgen

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target tissue and the impact of AR-mediated effects has been studied using human endometrial tissue samples and rodent models. In this review we highlight recent evidence that endometrial

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androgen biosynthesis and intracrine action is important in preparation of a tissue

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microenvironment that can support implantation and establishment of pregnancy. The impact of androgens on endometrial cell proliferation, in repair of the endometrial wound at the time of

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menstruation and in endometrial disorders is discussed. Future directions for research focused on AR function as a therapeutic target are considered.

35 Highlights

Androgens control key functional processes that regulate endometrial function.

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Androgen receptors are most abundant in stromal cells of normal endometrium and in

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epithelial cells of endometrial cancers. •

New evidence suggests both local activation and metabolism of androgens are essential for

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endometrial competence during the establishment of pregnancy. Dysregulation of androgen biosynthesis is associated with endometrial pathologies and

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impaired endometrial function. •

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Androgen receptors have important therapeutic potential for the regulation of endometrial function in health and disease.

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Androgens and endometrium. 1

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Introduction Androgens are present at high concentrations in the circulation of women and can act

directly on target tissues that express the androgen receptor (AR). Androgens have only

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recently emerged as important players in endometrial physiology supported by the

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demonstration that certain endometrial cell types express AR, with the protein subject to spatial and temporal regulation throughout the menstrual cycle. Recent evidence suggests that the endometrium also expresses enzymes capable of converting conjugated and other precursor steroids into testosterone (T) and dihydrotestosterone (DHT), and should therefore be considered as subject to intracrine androgen regulation. There is an increasing body of

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evidence to show that androgens can impact on endometrial proliferation, differentiation during the establishment of pregnancy and tissue repair during menstruation. Not surprisingly, dysregulation of androgen action in the endometrium is associated with endometrial pathologies, including endometriosis and endometrial cancer, and may also play an important

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role in infertility associated with endometrial dysfunction. The current review summarises our

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understanding to date of the impacts of androgen action in the endometrium and addresses the future potential for AR as a therapeutic target for the regulation of endometrial function in health

2 2.1

Structure of the endometrium and the human menstrual cycle Structure of the endometrium

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and disease.

The endometrium is a multicellular tissue whose primary function is to support a viable pregnancy and forms the inner lining of the uterus (Figure 1). It is divided into a basal layer and a luminal layer with an epithelium which establishes the tissue boundary to the uterine lumen. The stromal compartment consists of fibroblasts of mesenchymal origin and hosts a diverse

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group of immune cells sustained by a rich vascular supply, as well as epithelial cells that line the uterine lumen and the glands. The cells within the endometrium are sensitive to ovarian-derived steroids via extensive intercellular cross-talk which coordinates proliferation, differentiation,

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apoptosis and recruitment of cells in a spatiotemporal manner. Dysregulation of these processes results in endometrial-associated pathologies. A key feature of endometrial function

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in women and higher primates is the monthly breakdown and shedding of the luminal portion of the tissue during menstruation. Only one ‘menstruating’ mouse species has been described

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(Bellofiore, Ellery et al. 2017) while all other known mouse species only shed their endometrium following hormonal manipulation and endometrial stimulation to induce decidualisation followed by progesterone withdrawal (Kaitu'u-Lino, Morison et al. 2007, Cousins, Murray et al. 2014).

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Despite these differences, the conservation of uterine architecture between primates and

Endometrial responses to cyclical ovarian hormones

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rodents has provided a wealth of data of relevance to human reproduction and its disorders.

The human menstrual cycle lasts 28 days on average and is broadly defined by an oestrogen-dominated proliferative phase and a progesterone-dominated secretory phase.

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Unlike oestrogen and progesterone, circulating androgens do not fluctuate greatly across the menstrual cycle a mid-cycle peak of T at the time of ovulation has been reported (Salonia,

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Pontillo et al. 2008, Bui, Sluss et al. 2013).

The proliferative phase

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The proliferative phase is characterised by oestradiol (E2)-dependent cell proliferation and growth of stromal and epithelial compartments in response to rising circulating concentrations of

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E2 secreted by the ovarian follicles (Ferenczy 1976, Ludwig and Spornitz 1991). In the landmark study by Noyes et al, it was reported that during the proliferative phase it is the epithelial cells of the functional layer (luminal and glandular) that undergo rapid cell division so that the glands have a pseudo-stratified morphology and become more tortuous in the late proliferative phase

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(Noyes, Hertig et al. 1975).

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The secretory phase During the secretory phase, the endometrium transforms to prepare for pregnancy (Noyes,

Hertig et al. 1975). Stromal cell differentiation (decidualisation) is first detected in cells around

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the spiral arterioles and spreads throughout the whole functional compartment by the late

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secretory phase (de Ziegler, Fanchin et al. 1998). This transformation results in the formation of the ‘pre-decidua’, which is named to discriminate it from the true decidua, which is the maternal component of the placenta. Morphologically, this is characterised by stromal oedema and

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‘swelling’ of the stromal fibroblasts accompanied by an increase in nuclear size and cytoplasmic volume. Decidualisation occurs spontaneously in the absence of a blastocyst in species, such

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as humans, where the trophoblast of the early embryo breaches the luminal epithelium of the endometrium, with striking differences in the degree of decidualisation between primate species

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which reflects the degree of trophoblastic invasion (Ramsey, Houston et al. 1976). The decidua plays a dual role in producing factors that will promote blastocyst attachment and embryo

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growth, providing a foeto-maternal interface for oxygen supply to the embryo but also acts to limit the invasive properties of the trophoblast (Graham and Lala 1991). Progesterone plays a key role in stimulating stromal cells to decidualise acting via the progesterone receptor (PR). Decidualisation can be simulated in mice following priming with ovarian hormones and

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simulation of embryo implantation by directly stimulating the uterine epithelium (Ledford, Rankin et al. 1976, Brasted, White et al. 2003, Lee, Jeong et al. 2007, Cousins, Murray et al. 2014). A

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study using global PR knockout mice (targeted disruption of both PR-A and PR-B)

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demonstrated that they are unable to respond to an artificial decidualisation stimulus (Lydon, DeMayo et al. 1995), with human studies complementing animal model findings (Lessey, Yeh et

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al. 1996, Labied, Kajihara et al. 2006) and confirming a fundamental role for PR in the regulation of decidualisation.

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3 3.1

Expression of AR in the human and rodent endometrium Expression and regulation of AR in the endometrium of women Androgens have only recently emerged as important players in endometrial physiology.

Evidence that the endometrium is an androgen-target tissue is supported by detection of AR in the tissue. Early studies utilising dextran-coated charcoal adsorption followed by sucrose

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density centrifugation analysis of human endometrial homogenates revealed the existence of a protein capable of binding radiolabelled DHT (Tamaya, Motoyama et al. 1979, Muechler 1987). This protein was verified as AR and was subsequently detected in fixed tissue sections using immunohistochemistry and localised to nuclei in stromal, epithelial, endothelial and myometrial

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(smooth muscle) cells of the human uterus and decidua (Horie, Takakura et al. 1992, Mertens, Heineman et al. 1996, Mertens, Heineman et al. 2001, Milne, Henderson et al. 2005).

Real-time PCR quantifying AR mRNA in homogenates of human endometrial tissue

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samples obtained from different phases of the menstrual cycle showed highest expression during the proliferative phase and this significantly declined in the secretory phase (Vienonen,

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Miettinen et al. 2004). Detailed analysis of full thickness endometrial tissue sections has revealed intense AR immunostaining in stromal cell nuclei in both luminal and basal regions of the proliferative phase, while expression in the luminal cells declined during the secretory phase but was retained in the basal stromal cells (Marshall, Lowrey et al. 2011). Expression of AR in the luminal and glandular epithelial cells is significantly upregulated during the late secretory

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phase at a time when circulating progesterone concentrations decline (Taylor, Guzail et al. 2005, Marshall, Lowrey et al. 2011). Notably, expression of AR in endometrial glands, as detected by immunohistochemistry, is also increased following treatment with PR antagonists

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(Narvekar, Cameron et al. 2004).

Androgens regulate the expression of their receptor both at the mRNA and protein levels.

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Long term androgen administration to female-to-male transsexuals induces upregulation of AR in the uterine stroma and myometrium compared to non-treated individuals, suggesting a feedforward mechanism of androgen action in the endometrium (Chadha, Pache et al. 1994). Similarly, post-menopausal women administered with either E2 or a combination of E2 plus T for three months displayed a significant increase in AR immunoreactivity in the stroma after treatment with E2 plus T (Zang, Sahlin et al. 2008). It is noteworthy that in in the group treated

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with T alone, treatment induced endometrial atrophy in the post-menopausal endometrium to the extent that sample collection was compromised (Zang, Sahlin et al. 2008).

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Polycystic ovarian syndrome (PCOS) is often accompanied by hyperandrogenism and quantitative assessment of AR immunohistoscoring in the endometrium of women with PCOS has revealed a significant upregulation of AR protein in the glands during the proliferative and

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secretory phases and the stroma during the secretory phase of the cycle (Apparao, Lovely et al.

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2002). In the same study, Western blot analysis of Ishikawa cells (human endometrial epithelial adenocarcinoma cells) treated with or without DHT and/or the antiandrogen hydroxyflutamide demonstrated an androgen-dependent increase in total AR protein (Apparao, Lovely et al.

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2002).

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In the human endometrium, the temporal pattern of expression of AR described above

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would be consistent with upregulation by oestrogen and downregulation by progesterone (Mertens, Heineman et al. 1996, Mertens, Heineman et al. 2001). This is also supported by findings obtained in studies using human primary endometrial stromal fibroblasts treated with oestrogen in vitro, whereby a significant increase in T binding sites (equivalent to AR protein

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levels) and AR mRNA was reported, compared to controls (Fujimoto, Nishigaki et al. 1994,

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Fujimoto, Nishigaki et al. 1995). Burton et al, demonstrated that intra-uterine administration of the potent synthetic progestogen Levonorgestrel to women resulted in a dramatic timedependent decrease of AR mRNA and protein (Burton, Henderson et al. 2003). In contrast, the

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endometrium of women administered with the anti-progestin mifepristone exhibited elevated expression of AR protein in epithelial and stromal cells (Slayden, Nayak et al. 2001, Narvekar,

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Cameron et al. 2004). Thus, current evidence suggests that while androgens and oestrogens positively regulate AR expression, progestins downregulate AR in the endometrium.

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Expression and regulation of AR in the endometrium of rodents AR is expressed in the endometrium of rodents, with concordant expression of AR in

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stromal cells, however, there are conflicting reports regarding its expression in epithelial and endothelial cells (Hirai, Hirata et al. 1994, Pelletier, Labrie et al. 2000). Analysis by Northern blotting and in situ hybridisation reported detection of Ar mRNA throughout the mouse and rat endometrium, including the glandular epithelium (Hirai, Hirata et al. 1994, Pelletier 2000,

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Pelletier, Luu-The et al. 2004). In contrast, immunohistochemistry using mouse endometrium

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revealed intense staining in stromal fibroblasts, while epithelial cells were AR-negative (Xu, Li et al. 2015, Simitsidellis, Gibson et al. 2016). Data from our own studies confirm intense immunopositive staining of AR in stromal cell nuclei but failed to detect any AR-positive

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endometrial endothelial cells (Figure 2), a result that contrasts with data from our studies on human endometrium.

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In the mouse endometrium, like in the human, AR has been shown to be hormone-

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regulated. Specifically, ovariectomised mice treated with either E2 or DHT displayed an increase in AR immunoreactivity in the stroma, with DHT also inducing a striking increase of AR

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expression in the glandular and luminal epithelium (Xu, Li et al. 2015, Simitsidellis, Gibson et al. 2016). In contrast, treatment of ovariectomised mice with progesterone resulted in a reduction

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of AR staining, in agreement with the reported downregulation of AR during the secretory phase of the menstrual cycle (Xu, Li et al. 2015) and in response to synthetic progestins in women

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(Burton, Henderson et al. 2003).

Mouse models of uterine androgen action The Ar gene is located on the X chromosome (Chang, Kokontis et al. 1988, Lubahn,

Joseph et al. 1988) and the role of AR in the endometrium and some of its constituent cell types

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has been investigated using the CRE-LoxP system following introduction of LoxP sequences

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flanking exons encoding the AR DNA-binding domain (Yeh, Tsai et al. 2002, Schauwaers, De Gendt et al. 2007, Walters, Allan et al. 2007). Most of the studies in female mice have focused on ovarian function and have revealed subfertility phenotypes resulting from defective

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folliculogenesis or neuroendocrine defects ((Walters, McTavish et al. 2009), also reviewed by (Chang, Yeh et al. 2013)). A recent study by Walters and colleagues has used mice with global and cell-specific ablation of Ar combined with a DHT-induced model of PCOS to demonstrate that androgen actions in both neurones and granulosa cells are required for the development of both metabolic and reproductive phenotypes (Caldwell, Edwards et al. 2017). Global ARKO

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female mice (Ar-/-) display abnormal uterine growth with a significant reduction in uterine and endometrial surface area at diestrus and estrus compared to wild-type (WT) mice (Walters, McTavish et al. 2009). In addition, reciprocal ovarian transplantation experiments between WT and Ar-/- mice showed that ovariectomised Ar-/- hosts implanted with a WT ovary exhibited

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more pronounced defects in uterine growth than WT hosts with Ar-/- ovaries, suggesting a significant additional role for uterine AR in supporting fertility (Walters, McTavish et al. 2009). To date, no stroma-specific ARKO mouse model has been described, however a uterine gland-specific AR knockout (ugeARKO), generated using using the probasin (PBSN) promoter

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to target CRE expression to endometrial glands, identified a previously underappreciated ARdependent contribution of this compartment to uterine homeostasis (Choi, Zheng et al. 2015). Specifically, ovariectomised ugeARKO mice treated with T displayed a reduced increase in

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myometrial area compared to WT mice, suggesting an indirect effect of glandular AR action on the myometrium (Choi, Zheng et al. 2015). Our own studies support a role for AR-dependent regulation of the uterine glandular compartment, demonstrating DHT-dependent proliferation of

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uterine glandular epithelial cells and expansion of the glandular compartment in ovariectomised mice stimulated with DHT (Simitsidellis, Gibson et al. 2016). Mice expressing a transgenic luciferase reporter gene under the control of AR-specific

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response elements (ARE) have been used to demonstrate AR-dependent gene action in target organs (Dart, Waxman et al. 2013). Ligand-dependent transcriptional activation by AR, as

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measured by emitted bioluminescence and immunostaining of luciferase, was detected in the mouse uterus (Dart, Waxman et al. 2013). Moreover, treatment with the antiandrogen bicalutamide resulted in a significant reduction of detected bioluminescence, highlighting the utility of this mouse model for investigating AR modulation in vivo (Dart, Waxman et al. 2013). Taken together, these studies provide evidence for a functional role for AR expression and ARdependent signalling in the regulation of uterine function that offers key insights into the role of

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androgens in the regulation of endometrial physiology in women.

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Expression of androgen-metabolising enzymes and androgen synthesis in the endometrium. The classical view of endocrinology is that active steroid hormones synthesised in the

gonads and adrenal glands, alter the function of tissues, such as the endometrium, following

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delivery via the bloodstream. In 1988 the term ‘intracrine’ was defined by Fernand Labrie

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introducing the concept of concomitant steroid synthesis and action within the same cell (Labrie, Belanger et al. 1988), providing a novel mechanism which can lead to augmentation of endocrine and paracrine effects of steroids. Since then, an increasing number of tissues have

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been identified that may be subject to intracrine effects, as they express certain enzymes that metabolise steroids. These include the skin (Pomari, Dalla Valle et al. 2015), muscle (reviewed

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in (Yarrow, McCoy et al. 2012)) and fat (Dalla Valle, Toffolo et al. 2006).

In women, the most abundant circulating steroid is dehydroepiandrosterone (DHEA; together with its sulphated form DHEAS) which has weak affinity for AR but can act as precursor for the biosynthesis of androgens and oestrogens. The adrenal gland contributes more than 80% of circulating DHEA in women, with the ovaries contributing the other 20%

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(Labrie, Martel et al. 2011). The interconversion of DHEA and DHEAS is regulated by the action of steroid sulphotransferases (SULT2A1 and SULT2B1), which are responsible for the

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sulphation of DHEA and are expressed predominantly in the liver and the adrenal glands (LuuThe, Dufort et al. 1995, Geese and Raftogianis 2001), and steroid sulphatase, which is expressed in a multitude of tissues, albeit at low levels (Miki, Nakata et al. 2002, Suzuki, Miki et

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al. 2003). In women, circulating concentrations of DHEA (Labrie, Bélanger et al. 1997) and DHEAS (Labrie, Bélanger et al. 1997, Kiechl, Willeit et al. 2000) decline significantly with increasing age, although both the adrenals and the ovaries (albeit at much lower levels) retain the ability to synthesise DHEA after menopause. A direct correlation between the circulating

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levels of DHEA and the concentrations of eleven DHEA metabolites has led to reports suggesting adrenal DHEA is the predominant steroid precursor after menopause (Labrie, Martel et al. 2011).

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There is evidence from studies on human endometrial tissue for expression of several enzymes that play key roles in steroid biosynthesis and metabolism and which could influence

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tissue concentrations of androgens (Table 1). Specifically, the first and rate-limiting step in steroid synthesis involves the transport of cholesterol into the mitochondria by the steroidogenic

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acute regulatory protein (StAR) followed by the production of pregnenolone by the enzyme CYP11A1. Expression of STAR mRNA was detected at low levels in endometrial stromal cells but not epithelial cells, while StAR protein levels detected by Western blotting in total

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endometrial tissue homogenates were low to undetectable (Tsai, Wu et al. 2001, Aghajanova,

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Hamilton et al. 2009, Attar, Tokunaga et al. 2009). The transcript of CYP11A1 is detected in the normal endometrium at modest levels, but there are no human studies, to our knowledge,

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where CYP11A1 protein expression and enzymatic activity have been investigated (Hukkanen, Mäntylä et al. 1998, Rhee, Oh et al. 2003, Aghajanova, Hamilton et al. 2009, Luu-The 2013).

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Pregnenolone can be metabolised to either progesterone or via 17α-hydroxypregnenolone to DHEA by the actions of 3βHSDs (3βHSD1 and 3βHSD2) and CYP17A1 respectively. A study

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by Rhee and colleagues reported expression of 3βHSD1 mRNA and protein in total endometrial tissue homogenates and localised the protein to the glandular epithelium of proliferative and secretory phase endometrial samples (Rhee, Oh et al. 2003). Similarly, CYP17A1 transcripts were detected in the human endometrium, with no protein data available (Aghajanova, Hamilton

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et al. 2009, Chen, Saini et al. 2011, Huhtinen, Saloniemi-Heinonen et al. 2014). DHEA can be

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metabolised to androstenedione, a weak androgen with low affinity for AR, by 3βHSDs and after a series of enzymatic steps converted to the potent androgens T and DHT by the action of 17βHSDs and 5α-reductases (SRD5A1 and SRD5A2) respectively. In the normal human

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endometrium 17βHSD1, 17βHSD2 and SRD5A1 are detected both at the mRNA and protein levels (Ito, Suzuki et al. 2002, Ito, Utsunomiya et al. 2006, Rizner, Smuc et al. 2006, Dassen, Punyadeera et al. 2007, Carneiro, Morsch et al. 2008, Delvoux, Groothuis et al. 2009, Huhtinen, Saloniemi-Heinonen et al. 2014). Additional studies are required to elucidate the menstrual cycle-dependent expression of steroidogenic enzymes in the endometrium, since the majority of

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available data is derived either from pooled endometrial tissue of mixed proliferative and secretory phase or from isolated endometrial stromal cells.

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Endometrial androgen synthesis during the secretory phase and in preparation for pregnancy

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In preparation for pregnancy, the uterus undergoes differentiation and remodelling. A study in a mouse model of artificial decidualisation has demonstrated that the decidualised endometrium expresses all the transcripts of steroidogenic enzymes necessary for the

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formation of T (Star, Cyp11a1, Cyp17a1, Hsd3b, Hsd17b) (Das, Mantena et al. 2009). In addition, expression of 5α-reductase (Srd5a1) mRNA is significantly increased in the mouse

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uterus after treatment with oestrogen and progesterone, while 5α-reductase activity, as measured by the formation of DHT from radiolabelled T, is significantly increased in the gravid uterine horn of pregnant mice compared to the non-gravid horn (Minjarez, Konda et al. 2001). Women who had undergone fertility treatment with recombinant FSH and hCG, displayed a four-fold increase in STAR mRNA concentrations in their endometrium compared to early

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secretory phase endometrium of women without treatment (Macklon, van der Gaast et al. 2008). In addition, the transcript of CYP11A1 was detected by RT-PCR analysis in human decidua of early pregnancy and 3βHSD1 and 3βHSD2 were detected in human decidual tissue

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by immunohistochemistry (Rhee, Oh et al. 2003). A recent study from our group using in vitro decidualisation of human primary endometrial stromal cells has shown 3βHSD protein in intact

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cells by immunofluorescence and cell lysates by Western blotting, with no significant difference in levels between decidualised and control cells (Gibson, McInnes et al. 2013). Endometrial AKR1C3 (HSD17B5; preferentially converts androstenedione to T) mRNA concentrations are significantly higher during the early secretory phase of the menstrual cycle (Catalano, Wilson et al. 2011). Similarly, Huhtinen et al reported a trend for an increase of AKR1C3 mRNA expression in the secretory phase of the menstrual cycle compared to the

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proliferative phase from human endometrial tissue homogenates (Huhtinen, Desai et al. 2012). AKR1C3 protein was detected by immunohistochemistry mostly in epithelial and vascular cells

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(vascular smooth muscle and endothelial cells) with minimal to low expression in the stroma of secretory phase human endometria (Pelletier, Luu-The et al. 1999, Ito, Utsunomiya et al. 2006, Catalano, Wilson et al. 2011). However, in vitro decidualisation of primary human endometrial

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stromal cells (hESC) is accompanied by significantly increased expression of AKR1C3 at the

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mRNA and protein levels in a temporal manner and is associated with increased secretion of T in culture medium (Gibson, Simitsidellis et al. 2016). The same study demonstrated expression of SRD5A1 in decidualised hESC and secretion of DHT into culture medium (Gibson, Simitsidellis et al. 2016). T may also act as a precursor to biosynthesis of E2 in cells expressing CYP19A1 and decidualisation of hESC is also associated with increased expression of

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CYP19A1 protein, CYP19A1 activity and secretion of E2 into culture media (Gibson, McInnes et

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al. 2013).

As most of the genes encoding cytochrome P450 enzymes have cAMP response elements in their promoters (Payne, Youngblood et al. 1992, Youngblood and Payne 1992, Michael,

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Michael et al. 1997), it is perhaps not unexpected that their expression would be increased in

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response to rising cAMP production during decidualisation (for a review on cAMP regulation of cytochrome P-450 enzymes see (Lund, Zaphiropoulos et al. 1991)). Taken together, the above findings highlight that the endometrium gains steroidogenic capacity during preparation for

AR or as precursors for oestrogen biosynthesis (reviewed in (Gibson, Simitsidellis et al. 2016)).

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pregnancy and is characterized by local biosynthesis of androgens that can act both directly on

A role for androgens in regulation of endometrial decidualisation Studies described above demonstrate that decidualisation is associated with generation of

an androgen-rich microenvironment that can act regulate AR via endogenous signalling. Studies from rodent models targeting AR with exogenous ligands support key roles for AR-dependent

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regulation of decidualisation. Using a rat model, Chandrasekhar and colleagues showed that treatment with the anti-androgen hydroxyflutamide resulted in delayed blastocyst implantation and smaller foetuses on day 19 of pregnancy (Chandrasekhar, Armstrong et al. 1990). They

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attributed these effects to an anti-progestogenic effect of hydroxyflutamide (Chandrasekhar, Armstrong et al. 1990) but a year later they reported that the anti-progestogenic effect of

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hydroxyflutamide is not derived from its binding to PR but from a downregulation of oestrogeninduced PR expression (Chandrasekhar and Armstrong 1991), possibly via ER-AR-dependent

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cross-talk. In a mouse model of artificial decidualisation, it was shown that while T was unable to initiate the process (in the absence of progesterone), both T and DHT were able to maintain decidualisation from days 6-8 of treatment based on uterine weight and alkaline phosphatase

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activity, a marker of decidualisation (Zhang and Croy 1996). In an in vitro model, when primary

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mouse endometrial stromal cells treated with E2 and progesterone to induce decidualisation were co-stimulated with DHEA, they displayed a dose-dependent decrease in reactive oxygen

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species production following a H2O2 challenge (Qin, Qin et al. 2016). Subsequently Diao et al used a mouse model of delayed implantation to demonstrate that treatment with a low dose of

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testosterone propionate (TP) resulted in a longer time to implantation and a high dose of TP resulted in successful implantation but this was accompanied by abnormal expression of some

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decidual genes (Ptgs2, Lf) (Diao, Su et al. 2008). These studies highlight the potential for androgen availability to both positively and negatively impact on endometrial responses during the establishment of pregnancy.

Complementary studies have investigated the roles of T and DHT in fertility using primary

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hESC induced to decidualise in vitro. Cloke et al demonstrated that co-treatment of

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decidualising hESC with DHT results in AR-dependent increases in expression and secretion of the decidualisation makers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL) (Cloke, Huhtinen et al. 2008). Treatment of primary hESC with DHT during

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decidualisation also causes alterations in the shape index of cells, ultrastructural features including expanded endoplasmic reticulum,

increased numbers of mitochondria and lipid

droplets (Kajihara, Tanaka et al. 2014) and increased expression of the decidualisationassociated transcription factor FOXO1 (Kajihara, Tochigi et al. 2012). The same group reported that DHT significantly decreased H2O2-induced apoptosis in a dose-dependent manner

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accompanied by increased expression of the enzyme superoxide dismutase 2 (SOD2), which protects against oxidative stress (Kajihara, Tochigi et al. 2012). Notably, Cloke and colleagues reported that AR expression in hESC is decreased by progesterone treatment, however, decidualised hESC had increased androgen responsiveness due to a significant downregulation

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of AR SUMOylation by the E3 ligase PIAS1, resulting in enhanced AR transcriptional activity (Cloke, Huhtinen et al. 2008). In the same study, RNA silencing of AR identified a unique set of transcripts regulated by AR that were mainly associated with cytoskeletal organization (MCL2, WASPIP) and cell-cycle control (MCM4, WEE1) and that were distinct from those regulated by

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PR (Cloke, Huhtinen et al. 2008), highlighting a distinct role for AR in regulating transcriptional

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responses during decidualisation.

A role for androgens in endometrial wound healing

One of the most remarkable features of endometrial tissue is its ability to undergo sequential ‘wounding’ and rapid restoration of tissue integrity (healing) without developing a

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scar. Scarless tissue repair is rare in adult tissues, with the endometrium and oral mucosa being two prime examples. There are two main events when scar-free healing is required to maintain

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endometrial function. First, during each non-pregnant monthly menstrual cycle at the time of

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menstruation, and second, when the foetus is delivered (parturition). Defects in the process of endometrial wound healing during menstruation or miscarriage can contribute to heavy

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menstrual bleeding or intrauterine adhesions (Asherman’s syndrome) respectively (reviewed in (March 2011)). Deficits in postpartum repair are believed to predispose women to serious

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complications in subsequent pregnancies including placenta accreta (Fitzpatrick, Sellers et al. 2012).

Given that AR is expressed in the endometrium during the menstrual phase (Marshall, Lowrey et al. 2011), we and others have speculated that androgenic ligands might influence endometrial repair processes. At the time of menstruation, circulating blood concentrations of

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androstenedione and T remain relatively high at a time when oestrogen and progesterone concentrations have declined (Abraham 1974, Massafra, Gioia et al. 2000). Women with PCOS

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typically have elevated blood concentrations of androgens and in these women there have been reports of heavy or extended bleeding during menstruation (Maslyanskaya, Talib et al. 2017). In vitro assays using isolated hESC have identified that androgens regulate genes involved in

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regulation of apoptosis and cell migration, and functional studies using scratch assays have

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demonstrated that DHT can delay wound healing (Marshall, Lowrey et al. 2011). This hypothesis was also supported by data showing that androgens can have an impact of wound healing of the skin, a process which shares key features with endometrial repair, including infiltration of immune cells (Gilliver, Ashworth et al. 2006). For example, in rats, application of DHT retarded re-epithelialization of excisional and incisional skin wounds (Gilliver, Ruckshanthi

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et al. 2009), while in mice, topical application of an androgen receptor antagonist (Flutamide)

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improved closure of skin wounds (Toraldo, Bhasin et al. 2012).

To date there has been no information about the role of androgens in postpartum endometrial repair although our studies have implicated them in regulation of myometrial

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contractility (Makieva, Hutchinson et al. 2016). Although only women and primates menstruate,

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we (Cousins, Murray et al. 2014, Cousins, Murray et al. 2016) and others (Kaitu'u-Lino, Morison et al. 2007) have developed a mouse model of endometrial shedding/repair to simulate ‘menstruation’ and shown that it recapitulates key features of human menses, including

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synchronous shedding/repair (Garry, Hart et al. 2009), as well as rapid re-epithelialization. Using this model, we have investigated whether androgens might modulate endometrial ‘wound’

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repair (Cousins, Kirkwood et al. 2016). Administration of a single injection of DHT synchronously with induction of endometrial shedding and immune cell invasion demonstrated changes both in endometrial gene expression and tissue architecture as well as the overt appearance of blood in the vagina of the mice over 48 hours, which was more protracted

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compared to mice in which androgen was not administered (Cousins, Kirkwood et al. 2016). Examination of tissue slices from the endometrium of treated and untreated (DHT replete and DHT deprived) mice, showed clear histological changes and an apparent delay in the process of endometrial breakdown/shedding as well as the re-epithelialization of the endometrial surface in the treated group (see Figure 2 in (Cousins, Kirkwood et al. 2016)). Consistent with this impact

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of androgens, expression of genes associated with tissue remodeling, cell adhesion and ECM were altered by DHT treatment. Further analysis demonstrated that key genes, including matrix metalloproteinase 3 (Mmp3) and Snai3, a gene associated with mesenchymal-epithelial transition, both contain putative androgen response elements in their promoters (Cousins,

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Kirkwood et al. 2016). Immunohistochemistry of MMP3 revealed striking spatial and temporal changes in expression in response to DHT (Figure 3).

The impact of DHT on the expression of MMP is particularly interesting as it is wellestablished that these proteins play a critical role in endometrial breakdown and repair in

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women (Gaide Chevronnay, Selvais et al. 2012). Changes in the expression of MMP3 and MMP9 have also been identified as playing a role in wound healing of skin (Gill and Parks 2008) and androgens have been reported to alter expression of matrix proteins during experiments

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exploring hormonal regulation of cutaneous wound healing using a rat model (Gilliver, Ruckshanthi et al. 2007). Although steroidal regulation of MMPs is not a new concept, the studies described above highlight them as a direct target for androgen-dependent gene

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regulation in the endometrium.

Impact of androgens on endometrial proliferation

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A striking impact of androgens on endometrial function has been described in individuals undergoing sexual reassignment (female-to-male transition; FtM). Treatment for sex

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reassignment usually begins with T analogues that are prescribed for several months (usually more than a year and a maximum of 5 years) and there is cessation of menstruation usually

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within 5 months (Hembree, Cohen-Kettenis et al. 2009). The doses of T that are administered are within the range of 160-240 mg per day, with circulating concentrations being between 350700 ng/dl (Hembree, Cohen-Kettenis et al. 2009), which is equivalent to circulating T concentrations in men and far higher than in normal women. The majority of studies on the endometrium of women treated with T demonstrate atrophy

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of the tissue (Miller, Bedard et al. 1986), with reduced cell proliferation resulting in a tissue that resembles post-menopausal endometrium (Perrone, Cerpolini et al. 2009). Results from our

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own archived tissue are shown in Figure 4. Very few cases of gynaecologic malignancies occur in FtM individuals (Urban, Teng et al. 2011), which may be due to E2 suppression induced by T in this group (Perrone, Cerpolini et al. 2009). It must be noted that Futterweit et al reported that

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in their group of FtM individuals treated with T, the endometria appeared proliferative and in two

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instances cystic hyperplasia was detected (Futterweit and Deligdisch 1986). However, the only criterion they used for their study was histological examination and patient characterisation did not provide enough information to determine whether fat depots were large enough to increase peripheral biosynthesis of E2. In women and primates, administration of the anti-progestin mifepristone results in increased expression of AR in epithelial and stromal cells, an effect that

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is associated with anti-proliferative actions of this compound (Slayden, Nayak et al. 2001,

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Narvekar, Cameron et al. 2004). A recent study in which women were treated with a selective PR modulator (SPRM, Ulipristal acetate), a striking increase in AR immunoexpression was reported in glandular epithelial cells and low levels of proliferation (Whitaker, Murray et al.

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2017).

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The anti-proliferative effects of androgens in the human endometrium have also been demonstrated by in vitro studies using human primary endometrial cells (Tuckerman, Okon et al. 2000, Marshall, Lowrey et al. 2011). Tuckerman et al reported that treatment of human

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primary endometrial epithelial cells with the weak androgen androstenedione inhibited proliferation in a dose-dependent manner, with a significant reduction in 3H-thymidine uptake

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that was reversed by the AR antagonist cyproterone acetate (Tuckerman, Okon et al. 2000). This androstenedione-mediated effect was corroborated using an endometrial epithelial adenocarcinoma cell line (Ishikawa cells) (Park and Han 2013). That study showed that treatment of Ishikawa cells with increasing doses of androstenedione in vitro resulted in

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decreased cell proliferation and increased apoptosis and the authors argued for a direct effect of androstenedione, since they did not detect any changes in the expression of the enzymes 17βHSD1 and aromatase, which would have converted androstenedione to T and oestrone respectively (Park and Han 2013).

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Androgens also induce an anti-proliferative effect in decidualising hESC in vitro (Freis,

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Renke et al. 2017) and DHT reduces E2-induced proliferation of primary hESC isolated from proliferative phase endometrium (Marshall, Lowrey et al. 2011). We also have identified androgen-regulated genes using in silico approaches and validated their expression in primary

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hESC (Marshall, Lowrey et al. 2011). A recent study investigating expression of steroid hormone receptors in the cycling, menopausal, benign and malignant endometrium

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demonstrated that AR expression is retained in the post-menopausal endometrium and they suggested that loss of AR in endometrial cancer correlates with unfavourable prognosis and

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patient survival (Kamal, Bulmer et al. 2016). Taken together, these findings suggest that androgens have direct impacts on both epithelial and stromal cells in the endometrium, inducing

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an anti-proliferative effect, possibly via antagonising the effects of oestrogens. In contrast to the reported anti-proliferative endometrial effect of androgens in women, studies in rodents have demonstrated that androgens can have uterotrophic responses (Nantermet, Masarachia et al. 2005, Simitsidellis, Gibson et al. 2016). We recently described a

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distinct role for DHT in regulating uterine glandular growth and proliferation in ovariectomised mice. In this steroid-depleted model, treatment with DHT increased uterine weight, expanded

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the myometrial and endometrial area of the uterus and promoted uterine gland proliferation

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(Simitsidellis, Gibson et al. 2016). Impacts of DHT on proliferation were transient, peaking early followed by a refractory period similar to that observed with E2. These results complement those

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seen in the uterus of ARKO and ugeARKO mice, which exhibit defects in uterine growth and differentiation (as detailed above). Since most of the findings in the endometrium of androgen-

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treated

rodents

are

derived

from

studies

using

ovariectomised

animals

receiving

supraphysiological androgen doses, this could contribute to the observed discrepancy in androgen-mediated effects in rodents compared to humans. Evolutionary divergence between rodents and primates is reflected both in their capacity for steroidogenesis and adaptive physiology. For example, rodents lack adrenal CYP17A1 and therefore there is no contribution

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of DHEA or androstenedione from the rodent adrenal glands. Furthermore, in contrast to

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humans, rodents do not spontaneously decidualise and have multiple offspring per pregnancy, highlighting possible interspecies adaptations implicating androgen synthesis and signalling.

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Endometrial cancer

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9.1

Androgens and endometrial pathologies

Androgens may play a role in hormone-dependent cancers in women and androgen receptors have been proposed as potential therapeutic targets in breast, ovarian and

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endometrial cancers (reviewed in (Gibson, Simitsidellis et al. 2014, Chia, O'Brien et al. 2015)). Endometrial cancer (EC) is the fourth most common cancer in women in the developed world

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with incidence increasing in line with rates of obesity (reviewed in (Onstad, Schmandt et al. 2016)). In addition to obesity, exposure to unopposed oestrogens increases risk of aberrant endometrial proliferation and development of endometrial cancer (see (Sanderson, Critchley et al. 2017) for review of risk factors). A number of studies have assessed the link between exposure to endogenous hormones and endometrial cancer risk and reported evidence for an

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association between risk of developing EC and synthesis and action of androgens. Allen and colleagues measured concentrations of T, androstenedione and DHEAS in a large cohort of pre- and postmenopausal women in the European Prospective Investigation into Cancer and

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Nutrition (EPIC) study (Allen, Key et al. 2008). They reported that pre-diagnostic concentrations of total and free T were positively correlated with EC risk but that androstenedione and DHEAS

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were not associated with increased risk (Allen, Key et al. 2008). Recent assessment of circulating steroid hormones using highly sensitive liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS) and gas chromatography-MS methods in postmenopausal EC cases and healthy postmenopausal controls identified increased concentrations of DHEAS, DHEA, androstenedione and T in EC (Audet-Walsh, Lépine et al. 2011) providing strong evidence that increased serum androgens are associated with increased risk of EC in

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postmenopausal women. Consistent with this, there is an increased risk of EC in women with PCOS, which is associated with elevated circulating androgen concentrations (reviewed in

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(Barry, Azizia et al. 2014)). Furthermore, dysregulation of androgen biosynthetic enzymes has been reported in EC (recently reviewed in (Ito, Miki et al. 2016)). Androgen biosynthetic enzymes AKR1C3 and 5a-reductase are expressed in EC and studies suggest capacity for DHT

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synthesis within EC tumours (Tanaka, Miki et al. 2015).

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Extensive analysis of common genetic variation in 36 genes associated with sex steroid action reported genetic variation in AR was significantly associated with increased risk of EC (Yang, Gonzalez Bosquet et al. 2010). We and others have reported expression of AR in

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endometrial cancer, however reported AR expression within EC subtypes varies between studies. In our own assessment of endometrioid adenocarcinomas we have found consistent

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stromal and epithelial nuclear AR expression in well- and moderately differentiated cancers but AR expression is sporadic in poorly differentiated cancers that lack defined epithelial compartment (Gibson, Simitsidellis et al. 2014). A recent analysis of AR expression in 85 cases of EC as well as metastatic lesions, endometrial hyperplasia and normal postmenopausal

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control samples detected stromal and epithelial AR in all tissue types, with apparent loss of AR

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in high grade compared to low grade EC (Kamal, Bulmer et al. 2016). Recent data suggest AR could be a useful as a prognostic indicator in EC. Epithelial AR expression is associated with improved prognosis in EC (Kamal, Bulmer et al. 2016), while a high AR to ERα ratio is

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associated with poor survival (Tangen, Onyango et al. 2016). It has been proposed that targeting AR may be an effective strategy to impact on epithelial cell proliferation and limit the

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impact of increased exposure to androgen action through increased concentrations of circulating androgens, aberrant expression of steroid metabolising enzymes and mutations in the androgen receptor associated with increased risk of EC.

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Endometriosis Endometriosis is a chronic, incurable disorder with a complex pathophysiology

characterised by implantation of ectopic endometrial tissue within the peritoneal cavity (Giudice and Kao 2004). There is compelling evidence that androgens may have an impact on the pathophysiology of endometriosis. Androgens have an impact on processes implicated in

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establishment of endometriosis lesions (such as proliferation, tissue remodelling and repair; see

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above). We have shown that endometrial stromal fibroblasts in the basal zone of the endometrium remain AR-positive at time of menses (Marshall, Lowrey et al. 2011) and it is notable that fragments of basal endometrium are more common in menstrual effluent in women

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with endometriosis (Leyendecker, Herbertz et al. 2002). Furthermore, a study in which bioinformatics was used to integrate existing genomic datasets identified AR as a key

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endometriosis-associated transcription factor with 373 target genes of AR showing significant differential expression in endometriotic lesions compared to the normal endometrium (Yang,

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Kang et al. 2015). We have recently demonstrated that intracrine action of both oestrogens (Gibson, McInnes et al. 2013) and androgens (Gibson, Simitsidellis et al. 2016) is induced in

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decidualising endometrial stromal cells and others have reported that endometriosis lesions are characterised by high concentrations of testosterone, consistent with the creation of androgenrich microenvironment in endometriosis lesions (Huhtinen, Saloniemi-Heinonen et al. 2014). These data suggest that dysregulated androgen signalling may be present in endometriosis

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which may provide an effective therapeutic target. Indeed, early trials showed administration of the synthetic androgen Danazol (17alpha-ethinyl testosterone) reduced endometriosis lesion

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size. Despite its efficacy (Selak, Farquhar et al. 2007), Danazol is no longer recommended for

2014).

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treatment of endometriosis in Europe due to virilising side effects (Dunselman, Vermeulen et al.

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Summary and future perspectives The endometrium is an androgen target tissue and androgens control key functional

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processes that are required for endometrial function (Figure 5). AR is expressed in the endometrium and may be an important target in both physiological and pathophysiological contexts. New evidence suggests local activation and conversion of androgens is essential for endometrial competence during the establishment of pregnancy and that androgens are important regulators of decidualisation. The impact of androgens in the regulation of

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endometrial repair and proliferation suggests an important role for androgens in the regulation

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of the menstrual phase, a time when circulating oestrogens and progesterone are low. Recent insights suggest that androgens may have dual roles in the endometrium and may modulate endometrial function by acting directly on AR and indirectly as precursors to local oestrogens. Relative deficiency or excess of androgens can adversely impact on endometrial function and therefore dysregulation of androgen biosynthesis and action is associated with endometrial pathologies and impaired endometrial function.

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Androgen receptors have important therapeutic potential for the regulation of endometrial

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function in health and disease and understanding the discrete context-dependent mechanisms by which androgens regulate the endometrium is key to exploiting AR as a target. Consistent with other nuclear receptors, AR-dependent signalling can have tissue-specific effects and the

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impact on endometrial tissue can vary depending on cycle stage, menopausal status and circulating concentrations of androgens and androgen precursors. Androgens have proven efficacy for a number of different indications, however, the off-target actions of androgens that produce virilising side-effects have limited their use as a therapeutic in women. The development of a new class of compounds, selective androgen receptor modulators – SARMs

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(Gao and Dalton 2007, Burris, Solt et al. 2013), provides a timely opportunity to harness the potential beneficial impacts of androgen receptor therapy without the undesired side-effects.

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Importantly this class of compounds can have a spectrum of impacts on AR ranging from antagonist through to agonist profiles and understanding the potential impact of these new compounds on the endometrium in different physiological contexts will be fundamental to the

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appropriate application of these novel ligands and for the development of new therapies. Acknowledgements.

The research in the author’s laboratory is funded by the Medical Research Council (G1100356/1; MR/N024524/1 and MR/P00256X/1). The authors are grateful to Dr Alison

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Murray for providing tissue blocks of endometrium from women undergoing sex reversal held in local tissue archive.

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References

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Abraham, G. E. (1974). "Ovarian and adrenal contribution to peripheral androgens during the menstrual cycle." J Clin Endocrinol Metab 39(2): 340-346. Aghajanova, L., A. Hamilton, J. Kwintkiewicz, K. C. Vo and L. C. Giudice (2009). "Steroidogenic enzyme and key decidualization marker dysregulation in endometrial stromal cells from women with versus without endometriosis." Biol Reprod 80(1): 105114. Allen, N. E., T. J. Key, L. Dossus, S. Rinaldi, A. Cust, A. Lukanova, P. H. Peeters, N. C. OnlandMoret, P. H. Lahmann, F. Berrino, S. Panico, N. Larrañaga, G. Pera, M. J. Tormo, M. J. Sánchez, J. Ramón Quirós, E. Ardanaz, A. Tjønneland, A. Olsen, J. Chang-Claude, J. Linseisen, M. Schulz, H. Boeing, E. Lundin, D. Palli, K. Overvad, F. Clavel-Chapelon, M. C. Boutron-Ruault, S. Bingham, K. T. Khaw, H. B. Bueno-de-Mesquita, A. Trichopoulou, D. Trichopoulos, A. Naska, R. Tumino, E. Riboli and R. Kaaks (2008). "Endogenous sex hormones and endometrial cancer risk in women in the European Prospective Investigation into Cancer and Nutrition (EPIC)." Endocr Relat Cancer 15(2): 485-497. Apparao, K. B., L. P. Lovely, Y. Gui, R. A. Lininger and B. A. Lessey (2002). "Elevated endometrial androgen receptor expression in women with polycystic ovarian syndrome." Biol Reprod 66(2): 297-304. Attar, E., H. Tokunaga, G. Imir, M. B. Yilmaz, D. Redwine, M. Putman, B. Gurates, R. Attar, N. Yaegashi, D. B. Hales and S. E. Bulun (2009). "Prostaglandin E2 via steroidogenic factor-1 coordinately regulates transcription of steroidogenic genes necessary for estrogen synthesis in endometriosis." J Clin Endocrinol Metab 94(2): 623-631. Audet-Walsh, E., J. Lépine, J. Grégoire, M. Plante, P. Caron, B. Têtu, P. Ayotte, J. Brisson, L. Villeneuve, A. Bélanger and C. Guillemette (2011). "Profiling of endogenous estrogens, their precursors, and metabolites in endometrial cancer patients: association with risk and relationship to clinical characteristics." J Clin Endocrinol Metab 96(2): E330-339. Barry, J. A., M. M. Azizia and P. J. Hardiman (2014). "Risk of endometrial, ovarian and breast cancer in women with polycystic ovary syndrome: a systematic review and meta-analysis." Hum Reprod Update 20(5): 748-758. Bellofiore, N., S. J. Ellery, J. Mamrot, D. W. Walker, P. Temple-Smith and H. Dickinson (2017). "First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus)." Am J Obstet Gynecol 216(1): 40.e41-40.e11. Brasted, M., C. A. White, T. G. Kennedy and L. A. Salamonsen (2003). "Mimicking the events of menstruation in the murine uterus." Biol Reprod 69(4): 1273-1280. Bui, H. N., P. M. Sluss, S. Blincko, D. L. Knol, M. A. Blankenstein and A. C. Heijboer (2013). "Dynamics of serum testosterone during the menstrual cycle evaluated by daily measurements with an ID-LC-MS/MS method and a 2nd generation automated immunoassay." Steroids 78(1): 96-101. Bukulmez, O., D. B. Hardy, B. R. Carr, R. A. Word and C. R. Mendelson (2008). "Inflammatory status influences aromatase and steroid receptor expression in endometriosis." Endocrinology 149(3): 1190-1204. Burris, T. P., L. A. Solt, Y. Wang, C. Crumbley, S. Banerjee, K. Griffett, T. Lundasen, T. Hughes and D. J. Kojetin (2013). "Nuclear receptors and their selective pharmacologic modulators." Pharmacol Rev 65(2): 710-778. Burton, K. A., T. A. Henderson, S. G. Hillier, J. I. Mason, F. Habib, R. M. Brenner and H. O. Critchley (2003). "Local levonorgestrel regulation of androgen receptor and 17betahydroxysteroid dehydrogenase type 2 expression in human endometrium." Hum Reprod 18(12): 2610-2617.

RI PT

610

24


680

685

690

695

700

RI PT

SC

M AN U

675

TE D

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EP

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Caldwell, A. S. L., M. C. Edwards, R. Desai, M. Jimenez, R. B. Gilchrist, D. J. Handelsman and K. A. Walters (2017). "Neuroendocrine androgen action is a key extraovarian mediator in the development of polycystic ovary syndrome." Proc Natl Acad Sci U S A 114(16): E3334E3343. Carneiro, M. M., D. M. Morsch, A. F. Camargos, F. M. Reis and P. M. Spritzer (2008). "Androgen receptor and 5alpha-reductase are expressed in pelvic endometriosis." BJOG 115(1): 113-117. Catalano, R. D., M. R. Wilson, S. C. Boddy and H. N. Jabbour (2011). "Comprehensive expression analysis of prostanoid enzymes and receptors in the human endometrium across the menstrual cycle." Mol Hum Reprod 17(3): 182-192. Chadha, S., T. D. Pache, J. M. Huikeshoven, A. O. Brinkmann and T. H. van der Kwast (1994). "Androgen receptor expression in human ovarian and uterine tissue of long-term androgen-treated transsexual women." Hum Pathol 25(11): 1198-1204. Chandrasekhar, Y. and D. T. Armstrong (1991). "Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide." Biol Reprod 45(1): 78-81. Chandrasekhar, Y., D. T. Armstrong and T. G. Kennedy (1990). "Implantation delay and antideciduogenic activity in the rat by the anti-androgen, hydroxyflutamide." Biol Reprod 42(1): 120-125. Chang, C., S. Yeh, S. O. Lee and T. M. Chang (2013). "Androgen receptor (AR) pathophysiological roles in androgen-related diseases in skin, bone/muscle, metabolic syndrome and neuron/immune systems: lessons learned from mice lacking AR in specific cells." Nucl Recept Signal 11: e001. Chang, C. S., J. Kokontis and S. T. Liao (1988). "Molecular cloning of human and rat complementary DNA encoding androgen receptors." Science 240(4850): 324-326. Chen, Y., S. Saini, M. S. Zaman, H. Hirata, V. Shahryari, G. Deng and R. Dahiya (2011). "Cytochrome P450 17 (CYP17) is involved in endometrial cancinogenesis through apoptosis and invasion pathways." Mol Carcinog 50(1): 16-23. Chia, K., M. O'Brien, M. Brown and E. Lim (2015). "Targeting the androgen receptor in breast cancer." Curr Oncol Rep 17(2): 4. Choi, J. P., Y. Zheng, K. A. Skulte, D. J. Handelsman and U. Simanainen (2015). "Development and characterization of uterine glandular epithelium specific androgen receptor knockout mouse model." Biol Reprod. Cloke, B., K. Huhtinen, L. Fusi, T. Kajihara, M. Yliheikkila, K. K. Ho, G. Teklenburg, S. Lavery, M. C. Jones, G. Trew, J. J. Kim, E. W. Lam, J. E. Cartwright, M. Poutanen and J. J. Brosens (2008). "The androgen and progesterone receptors regulate distinct gene networks and cellular functions in decidualizing endometrium." Endocrinology 149(9): 4462-4474. Cousins, F. L., P. M. Kirkwood, A. A. Murray, F. Collins, D. A. Gibson and P. T. Saunders (2016). "Androgens regulate scarless repair of the endometrial "wound" in a mouse model of menstruation." FASEB J 30(8): 2802-2811. Cousins, F. L., P. M. Kirkwood, P. T. Saunders and D. A. Gibson (2016). "Evidence for a dynamic role for mononuclear phagocytes during endometrial repair and remodelling." Sci Rep 6: 36748. Cousins, F. L., A. Murray, A. Esnal, D. A. Gibson, H. O. Critchley and P. T. Saunders (2014). "Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation." PLoS One 9(1): e86378. Cousins, F. L., A. A. Murray, J. P. Scanlon and P. T. Saunders (2016). "Hypoxyprobe reveals dynamic spatial and temporal changes in hypoxia in a mouse model of endometrial breakdown and repair." BMC Res Notes 9(1): 30.

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Dalla Valle, L., V. Toffolo, A. Nardi, C. Fiore, P. Bernante, R. Di Liddo, P. P. Parnigotto and L. Colombo (2006). "Tissue-specific transcriptional initiation and activity of steroid sulfatase complementing dehydroepiandrosterone sulfate uptake and intracrine steroid activations in human adipose tissue." J Endocrinol 190(1): 129-139. Dart, D. A., J. Waxman, E. O. Aboagye and C. L. Bevan (2013). "Visualising androgen receptor activity in male and female mice." PLoS One 8(8): e71694. Das, A., S. R. Mantena, A. Kannan, D. B. Evans, M. K. Bagchi and I. C. Bagchi (2009). "De novo synthesis of estrogen in pregnant uterus is critical for stromal decidualization and angiogenesis." Proc Natl Acad Sci U S A 106(30): 12542-12547. Dassen, H., C. Punyadeera, R. Kamps, B. Delvoux, A. Van Langendonckt, J. Donnez, B. Husen, H. Thole, G. Dunselman and P. Groothuis (2007). "Estrogen metabolizing enzymes in endometrium and endometriosis." Hum Reprod 22(12): 3148-3158. de Ziegler, D., R. Fanchin, B. de Moustier and C. Bulletti (1998). "The hormonal control of endometrial receptivity: estrogen (E2) and progesterone." J Reprod Immunol 39(1-2): 149166. Delvoux, B., P. Groothuis, T. D'Hooghe, C. Kyama, G. Dunselman and A. Romano (2009). "Increased production of 17beta-estradiol in endometriosis lesions is the result of impaired metabolism." J Clin Endocrinol Metab 94(3): 876-883. Diao, H. L., R. W. Su, H. N. Tan, S. J. Li, W. Lei, W. B. Deng and Z. M. Yang (2008). "Effects of androgen on embryo implantation in the mouse delayed-implantation model." Fertil Steril 90(4 Suppl): 1376-1383. Dunselman, G. A., N. Vermeulen, C. Becker, C. Calhaz-Jorge, T. D'Hooghe, B. De Bie, O. Heikinheimo, A. W. Horne, L. Kiesel, A. Nap, A. Prentice, E. Saridogan, D. Soriano, W. Nelen and E. S. o. H. R. a. Embryology (2014). "ESHRE guideline: management of women with endometriosis." Hum Reprod 29(3): 400-412. Ferenczy, A. (1976). "Studies on the cytodynamics of human endometrial regeneration. I. Scanning electron microscopy." Am J Obstet Gynecol 124(1): 64-74. Fitzpatrick, K. E., S. Sellers, P. Spark, J. J. Kurinczuk, P. Brocklehurst and M. Knight (2012). "Incidence and risk factors for placenta accreta/increta/percreta in the UK: a national casecontrol study." PLoS One 7(12): e52893. Freis, A., T. Renke, U. Kämmerer, J. Jauckus, T. Strowitzki and A. Germeyer (2017). "Effects of a hyperandrogenaemic state on the proliferation and decidualization potential in human endometrial stromal cells." Arch Gynecol Obstet 295(4): 1005-1013. Fujimoto, J., M. Nishigaki, M. Hori, S. Ichigo, T. Itoh and T. Tamaya (1994). "The effect of estrogen and androgen on androgen receptors and mRNA levels in uterine leiomyoma, myometrium and endometrium of human subjects." J Steroid Biochem Mol Biol 50(3-4): 137-143. Fujimoto, J., M. Nishigaki, M. Hori, S. Ichigo, S. Morishita and T. Tamaya (1995). "Effects of Estradiol and Testosterone on the Synthesis, Expression and Degradation of Androgen Receptor in Human Uterine Endometrial Fibroblasts." J Biomed Sci 2(2): 160-165. Futterweit, W. and L. Deligdisch (1986). "Histopathological effects of exogenously administered testosterone in 19 female to male transsexuals." J Clin Endocrinol Metab 62(1): 16-21. Gaide Chevronnay, H. P., C. Selvais, H. Emonard, C. Galant, E. Marbaix and P. Henriet (2012). "Regulation of matrix metalloproteinases activity studied in human endometrium as a paradigm of cyclic tissue breakdown and regeneration." Biochim Biophys Acta 1824(1): 146-156. Gao, W. and J. T. Dalton (2007). "Expanding the therapeutic use of androgens via selective androgen receptor modulators (SARMs)." Drug Discov Today 12(5-6): 241-248.

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Garry, R., R. Hart, K. A. Karthigasu and C. Burke (2009). "A re-appraisal of the morphological changes within the endometrium during menstruation: a hysteroscopic, histological and scanning electron microscopic study." Hum Reprod 24(6): 1393-1401. Geese, W. J. and R. B. Raftogianis (2001). "Biochemical characterization and tissue distribution of human SULT2B1." Biochem Biophys Res Commun 288(1): 280-289. Gibson, D. A., K. J. McInnes, H. O. Critchley and P. T. Saunders (2013). "Endometrial Intracrinology--generation of an estrogen-dominated microenvironment in the secretory phase of women." J Clin Endocrinol Metab 98(11): E1802-1806. Gibson, D. A., I. Simitsidellis, F. Collins and P. T. Saunders (2014). "Evidence of androgen action in endometrial and ovarian cancers." Endocr Relat Cancer 21(4): T203-218. Gibson, D. A., I. Simitsidellis, F. L. Cousins, H. O. Critchley and P. T. Saunders (2016). "Intracrine Androgens Enhance Decidualization and Modulate Expression of Human Endometrial Receptivity Genes." Sci Rep 6: 19970. Gibson, D. A., I. Simitsidellis and P. T. Saunders (2016). "Regulation of androgen action during establishment of pregnancy." J Mol Endocrinol 57(1): R35-47. Gill, S. E. and W. C. Parks (2008). "Metalloproteinases and their inhibitors: regulators of wound healing." Int J Biochem Cell Biol 40(6-7): 1334-1347. Gilliver, S. C., J. J. Ashworth, S. J. Mills, M. J. Hardman and G. S. Ashcroft (2006). "Androgens modulate the inflammatory response during acute wound healing." J Cell Sci 119(Pt 4): 722-732. Gilliver, S. C., J. P. Ruckshanthi, S. J. Atkinson and G. S. Ashcroft (2007). "Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing." Lab Invest 87(9): 871-881. Gilliver, S. C., J. P. Ruckshanthi, M. J. Hardman, L. A. Zeef and G. S. Ashcroft (2009). "5alphadihydrotestosterone (DHT) retards wound closure by inhibiting re-epithelialization." J Pathol 217(1): 73-82. Giudice, L. C. and L. C. Kao (2004). "Endometriosis." Lancet 364(9447): 1789-1799. Graham, C. H. and P. K. Lala (1991). "Mechanism of control of trophoblast invasion in situ." J Cell Physiol 148(2): 228-234. Hembree, W. C., P. Cohen-Kettenis, H. A. Delemarre-van de Waal, L. J. Gooren, W. J. Meyer, N. P. Spack, V. Tangpricha, V. M. Montori and E. Society (2009). "Endocrine treatment of transsexual persons: an Endocrine Society clinical practice guideline." J Clin Endocrinol Metab 94(9): 3132-3154. Hirai, M., S. Hirata, T. Osada, K. Hagihara and J. Kato (1994). "Androgen receptor mRNA in the rat ovary and uterus." J Steroid Biochem Mol Biol 49(1): 1-7. Horie, K., K. Takakura, K. Imai, S. Liao and T. Mori (1992). "Immunohistochemical localization of androgen receptor in the human endometrium, decidua, placenta and pathological conditions of the endometrium." Hum Reprod 7(10): 1461-1466. Huhtinen, K., R. Desai, M. Stahle, A. Salminen, D. J. Handelsman, A. Perheentupa and M. Poutanen (2012). "Endometrial and endometriotic concentrations of estrone and estradiol are determined by local metabolism rather than circulating levels." J Clin Endocrinol Metab 97(11): 4228-4235. Huhtinen, K., T. Saloniemi-Heinonen, P. Keski-Rahkonen, R. Desai, D. Laajala, M. Stahle, M. R. Hakkinen, M. Awosanya, P. Suvitie, H. Kujari, T. Aittokallio, D. J. Handelsman, S. Auriola, A. Perheentupa and M. Poutanen (2014). "Intra-tissue steroid profiling indicates differential progesterone and testosterone metabolism in the endometrium and endometriosis lesions." J Clin Endocrinol Metab 99(11): E2188-2197. Hukkanen, J., M. Mantyla, L. Kangas, P. Wirta, J. Hakkola, P. Paakki, S. Evisalmi, O. Pelkonen and H. Raunio (1998). "Expression of cytochrome P450 genes encoding enzymes active in

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the metabolism of tamoxifen in human uterine endometrium." Pharmacol Toxicol 82(2): 93-97. Hukkanen, J., M. Mäntylä, L. Kangas, P. Wirta, J. Hakkola, P. Paakki, S. Evisalmi, O. Pelkonen and H. Raunio (1998). "Expression of cytochrome P450 genes encoding enzymes active in the metabolism of tamoxifen in human uterine endometrium." Pharmacol Toxicol 82(2): 93-97. Ito, K., Y. Miki, T. Suzuki, K. M. McNamara and H. Sasano (2016). "In situ androgen and estrogen biosynthesis in endometrial cancer: focus on androgen actions and intratumoral production." Endocr Relat Cancer 23(7): R323-335. Ito, K., T. Suzuki, J. Akahira, T. Moriya, C. Kaneko, H. Utsunomiya, N. Yaegashi, K. Okamura and H. Sasano (2002). "Expression of androgen receptor and 5alpha-reductases in the human normal endometrium and its disorders." Int J Cancer 99(5): 652-657. Ito, K., H. Utsunomiya, T. Suzuki, S. Saitou, J. Akahira, K. Okamura, N. Yaegashi and H. Sasano (2006). "17Beta-hydroxysteroid dehydrogenases in human endometrium and its disorders." Mol Cell Endocrinol 248(1-2): 136-140. Kaitu'u-Lino, T. J., N. B. Morison and L. A. Salamonsen (2007). "Estrogen is not essential for full endometrial restoration after breakdown: lessons from a mouse model." Endocrinology 148(10): 5105-5111. Kajihara, T., K. Tanaka, T. Oguro, H. Tochigi, J. Prechapanich, S. Uchino, A. Itakura, S. Sucurovic, K. Murakami, J. J. Brosens and O. Ishihara (2014). "Androgens modulate the morphological characteristics of human endometrial stromal cells decidualized in vitro." Reprod Sci 21(3): 372-380. Kajihara, T., H. Tochigi, J. Prechapanich, S. Uchino, A. Itakura, J. J. Brosens and O. Ishihara (2012). "Androgen signaling in decidualizing human endometrial stromal cells enhances resistance to oxidative stress." Fertil Steril 97(1): 185-191. Kamal, A. M., J. N. Bulmer, S. B. DeCruze, H. F. Stringfellow, P. Martin-Hirsch and D. K. Hapangama (2016). "Androgen receptors are acquired by healthy postmenopausal endometrial epithelium and their subsequent loss in endometrial cancer is associated with poor survival." Br J Cancer 114(6): 688-696. Kiechl, S., J. Willeit, E. Bonora, S. Schwarz and Q. Xu (2000). "No association between dehydroepiandrosterone sulfate and development of atherosclerosis in a prospective population study (Bruneck Study)." Arterioscler Thromb Vasc Biol 20(4): 1094-1100. Labied, S., T. Kajihara, P. A. Madureira, L. Fusi, M. C. Jones, J. M. Higham, R. Varshochi, J. M. Francis, G. Zoumpoulidou, A. Essafi, S. Fernandez de Mattos, E. W. Lam and J. J. Brosens (2006). "Progestins regulate the expression and activity of the forkhead transcription factor FOXO1 in differentiating human endometrium." Mol Endocrinol 20(1): 35-44. Labrie, C., A. Belanger and F. Labrie (1988). "Androgenic activity of dehydroepiandrosterone and androstenedione in the rat ventral prostate." Endocrinology 123(3): 1412-1417. Labrie, F., A. Bélanger, L. Cusan, J. L. Gomez and B. Candas (1997). "Marked decline in serum concentrations of adrenal C19 sex steroid precursors and conjugated androgen metabolites during aging." J Clin Endocrinol Metab 82(8): 2396-2402. Labrie, F., C. Martel and J. Balser (2011). "Wide distribution of the serum dehydroepiandrosterone and sex steroid levels in postmenopausal women: role of the ovary?" Menopause 18(1): 30-43. Ledford, B. E., J. C. Rankin, R. R. Markwald and B. Baggett (1976). "Biochemical and morphological changes following artificially stimulated decidualization in the mouse uterus." Biol Reprod 15(4): 529-535.

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ACCEPTED MANUSCRIPT

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Lee, K. Y., J. W. Jeong, S. Y. Tsai, J. P. Lydon and F. J. DeMayo (2007). "Mouse models of implantation." Trends Endocrinol Metab 18(6): 234-239. Lessey, B. A., I. Yeh, A. J. Castelbaum, M. A. Fritz, A. O. Ilesanmi, P. Korzeniowski, J. Sun and K. Chwalisz (1996). "Endometrial progesterone receptors and markers of uterine receptivity in the window of implantation." Fertil Steril 65(3): 477-483. Leyendecker, G., M. Herbertz, G. Kunz and G. Mall (2002). "Endometriosis results from the dislocation of basal endometrium." Hum Reprod 17(10): 2725-2736. Lubahn, D. B., D. R. Joseph, P. M. Sullivan, H. F. Willard, F. S. French and E. M. Wilson (1988). "Cloning of human androgen receptor complementary DNA and localization to the X chromosome." Science 240(4850): 327-330. Ludwig, H. and U. M. Spornitz (1991). "Microarchitecture of the human endometrium by scanning electron microscopy: menstrual desquamation and remodeling." Ann N Y Acad Sci 622: 28-46. Lund, J., P. G. Zaphiropoulos, A. Mode, M. Warner and J. A. Gustafsson (1991). "Hormonal regulation of cytochrome P-450 gene expression." Adv Pharmacol 22: 325-354. Luu-The, V. (2013). "Assessment of steroidogenesis and steroidogenic enzyme functions." J Steroid Biochem Mol Biol 137: 176-182. Luu-The, V., I. Dufort, N. Paquet, G. Reimnitz and F. Labrie (1995). "Structural characterization and expression of the human dehydroepiandrosterone sulfotransferase gene." DNA Cell Biol 14(6): 511-518. Lydon, J. P., F. J. DeMayo, C. R. Funk, S. K. Mani, A. R. Hughes, C. A. Montgomery, Jr., G. Shyamala, O. M. Conneely and B. W. O'Malley (1995). "Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities." Genes Dev 9(18): 2266-2278. Macklon, N. S., M. H. van der Gaast, A. Hamilton, B. C. Fauser and L. C. Giudice (2008). "The impact of ovarian stimulation with recombinant FSH in combination with GnRH antagonist on the endometrial transcriptome in the window of implantation." Reprod Sci 15(4): 357365. Makieva, S., L. J. Hutchinson, S. P. Rajagopal, S. F. Rinaldi, P. Brown, P. T. Saunders and J. E. Norman (2016). "Androgen-induced relaxation of uterine myocytes is mediated by blockade of both Ca flux and MLC phosphorylation." J Clin Endocrinol Metab: jc20152851. March, C. M. (2011). "Asherman's syndrome." Semin Reprod Med 29(2): 83-94. Marshall, E., J. Lowrey, S. MacPherson, J. A. Maybin, F. Collins, H. O. Critchley and P. T. Saunders (2011). "In silico analysis identifies a novel role for androgens in the regulation of human endometrial apoptosis." J Clin Endocrinol Metab 96(11): E1746-1755. Maslyanskaya, S., H. J. Talib, J. L. Northridge, A. M. Jacobs, C. Coble and S. M. Coupey (2017). "Polycystic Ovary Syndrome: An Under-recognized Cause of Abnormal Uterine Bleeding in Adolescents Admitted to a Children's Hospital." J Pediatr Adolesc Gynecol 30(3): 349-355. Massafra, C., D. Gioia, C. De Felice, E. Picciolini, V. De Leo, M. Bonifazi and A. Bernabei (2000). "Effects of estrogens and androgens on erythrocyte antioxidant superoxide dismutase, catalase and glutathione peroxidase activities during the menstrual cycle." J Endocrinol 167(3): 447-452. Mertens, H. J., M. J. Heineman, J. Koudstaal, P. Theunissen and J. L. Evers (1996). "Androgen receptor content in human endometrium." Eur J Obstet Gynecol Reprod Biol 70(1): 11-13. Mertens, H. J., M. J. Heineman, P. H. Theunissen, F. H. de Jong and J. L. Evers (2001). "Androgen, estrogen and progesterone receptor expression in the human uterus during the menstrual cycle." Eur J Obstet Gynecol Reprod Biol 98(1): 58-65. Michael, M. D., L. F. Michael and E. R. Simpson (1997). "A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene." Mol Cell Endocrinol 134(2): 147-156.

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ACCEPTED MANUSCRIPT

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Miki, Y., T. Nakata, T. Suzuki, A. D. Darnel, T. Moriya, C. Kaneko, K. Hidaka, Y. Shiotsu, H. Kusaka and H. Sasano (2002). "Systemic distribution of steroid sulfatase and estrogen sulfotransferase in human adult and fetal tissues." J Clin Endocrinol Metab 87(12): 57605768. Miller, N., Y. C. Bedard, N. B. Cooter and D. L. Shaul (1986). "Histological changes in the genital tract in transsexual women following androgen therapy." Histopathology 10(7): 661-669. Milne, S. A., T. A. Henderson, R. W. Kelly, P. T. Saunders, D. T. Baird and H. O. Critchley (2005). "Leukocyte populations and steroid receptor expression in human first-trimester decidua; regulation by antiprogestin and prostaglandin E analog." J Clin Endocrinol Metab 90(7): 4315-4321. Minjarez, D., V. Konda and R. A. Word (2001). "Regulation of uterine 5 alpha-reductase type 1 in mice." Biol Reprod 65(5): 1378-1382. Muechler, E. K. (1987). "The androgen receptor of the human endometrium." Endocr Res 13(1): 69-84. Nantermet, P. V., P. Masarachia, M. A. Gentile, B. Pennypacker, J. Xu, D. Holder, D. Gerhold, D. Towler, A. Schmidt, D. B. Kimmel, L. P. Freedman, S. Harada and W. J. Ray (2005). "Androgenic induction of growth and differentiation in the rodent uterus involves the modulation of estrogen-regulated genetic pathways." Endocrinology 146(2): 564-578. Narvekar, N., S. Cameron, H. O. Critchley, S. Lin, L. Cheng and D. T. Baird (2004). "Low-dose mifepristone inhibits endometrial proliferation and up-regulates androgen receptor." J Clin Endocrinol Metab 89(5): 2491-2497. Noyes, R. W., A. T. Hertig and J. Rock (1975). "Dating the endometrial biopsy." Am J Obstet Gynecol 122(2): 262-263. Onstad, M. A., R. E. Schmandt and K. H. Lu (2016). "Addressing the Role of Obesity in Endometrial Cancer Risk, Prevention, and Treatment." J Clin Oncol 34(35): 4225-4230. Park, S. B. and M. Han (2013). "Inhibitory effects of androstenedione on endometrial cells: implications for poor reproductive outcome among women with androgen excess." Eur J Obstet Gynecol Reprod Biol 171(2): 295-300. Payne, A. H., G. L. Youngblood, L. Sha, M. Burgos-Trinidad and S. H. Hammond (1992). "Hormonal regulation of steroidogenic enzyme gene expression in Leydig cells." J Steroid Biochem Mol Biol 43(8): 895-906. Pelletier, G. (2000). "Localization of androgen and estrogen receptors in rat and primate tissues." Histol Histopathol 15(4): 1261-1270. Pelletier, G., C. Labrie and F. Labrie (2000). "Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs." J Endocrinol 165(2): 359-370. Pelletier, G., V. Luu-The, S. Li and F. Labrie (2004). "Localization and estrogenic regulation of androgen receptor mRNA expression in the mouse uterus and vagina." J Endocrinol 180(1): 77-85. Pelletier, G., V. Luu-The, B. Têtu and F. Labrie (1999). "Immunocytochemical localization of type 5 17beta-hydroxysteroid dehydrogenase in human reproductive tissues." J Histochem Cytochem 47(6): 731-738. Perrone, A. M., S. Cerpolini, N. C. Maria Salfi, C. Ceccarelli, L. B. De Giorgi, G. Formelli, P. Casadio, T. Ghi, G. Pelusi, C. Pelusi and M. C. Meriggiola (2009). "Effect of long-term testosterone administration on the endometrium of female-to-male (FtM) transsexuals." J Sex Med 6(11): 3193-3200.

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Pomari, E., L. Dalla Valle, P. Pertile, L. Colombo and M. J. Thornton (2015). "Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts." FASEB J 29(2): 508-524. Qin, A., J. Qin, Y. Jin, W. Xie, L. Fan, L. Jiang and F. Mo (2016). "DHEA improves the antioxidant capacity of endometrial stromal cells and improves endometrium receptivity via androgen receptor." Eur J Obstet Gynecol Reprod Biol 198: 120-126. Ramsey, E. M., M. L. Houston and J. W. Harris (1976). "Interactions of the trophoblast and maternal tissues in three closely related primate species." Am J Obstet Gynecol 124(6): 647-652. Rhee, H. S., S. H. Oh, B. J. Ko, D. M. Han, B. H. Jeon, H. Park, H. B. Moon and W. S. Kim (2003). "Expression of 3beta-hydroxysteroid dehydrogenase and P450 side chain cleavage enzyme in the human uterine endometrium." Exp Mol Med 35(3): 160-166. Rizner, T. L., T. Smuc, R. Rupreht, J. Sinkovec and T. M. Penning (2006). "AKR1C1 and AKR1C3 may determine progesterone and estrogen ratios in endometrial cancer." Mol Cell Endocrinol 248(1-2): 126-135. Salonia, A., M. Pontillo, R. E. Nappi, G. Zanni, F. Fabbri, M. Scavini, R. Daverio, A. Gallina, P. Rigatti, E. Bosi, P. A. Bonini and F. Montorsi (2008). "Menstrual cycle-related changes in circulating androgens in healthy women with self-reported normal sexual function." J Sex Med 5(4): 854-863. Sanderson, P. A., H. O. Critchley, A. R. Williams, M. J. Arends and P. T. Saunders (2017). "New concepts for an old problem: the diagnosis of endometrial hyperplasia." Hum Reprod Update 23(2): 232-254. Schauwaers, K., K. De Gendt, P. T. Saunders, N. Atanassova, A. Haelens, L. Callewaert, U. Moehren, J. V. Swinnen, G. Verhoeven, G. Verrijdt and F. Claessens (2007). "Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model." Proc Natl Acad Sci U S A 104(12): 4961-4966. Selak, V., C. Farquhar, A. Prentice and A. Singla (2007). "Danazol for pelvic pain associated with endometriosis." Cochrane Database Syst Rev(4): CD000068. Simitsidellis, I., D. A. Gibson, F. L. Cousins, A. Esnal-Zufiaurre and P. T. Saunders (2016). "A Role for Androgens in Epithelial Proliferation and Formation of Glands in the Mouse Uterus." Endocrinology: en20152032. Slayden, O. D., N. R. Nayak, K. A. Burton, K. Chwalisz, S. T. Cameron, H. O. Critchley, D. T. Baird and R. M. Brenner (2001). "Progesterone antagonists increase androgen receptor expression in the rhesus macaque and human endometrium." J Clin Endocrinol Metab 86(6): 2668-2679. Suzuki, T., Y. Miki, T. Nakata, Y. Shiotsu, S. Akinaga, K. Inoue, T. Ishida, M. Kimura, T. Moriya and H. Sasano (2003). "Steroid sulfatase and estrogen sulfotransferase in normal human tissue and breast carcinoma." J Steroid Biochem Mol Biol 86(3-5): 449-454. Tamaya, T., T. Motoyama, Y. Ohono, N. Ide, T. Tsurusaki and H. Okada (1979). "Steroid receptor levels and histology of endometriosis and adenomyosis." Fertil Steril 31(4): 396400. Tanaka, S., Y. Miki, C. Hashimoto, K. Takagi, Z. Doe, B. Li, N. Yaegashi, T. Suzuki and K. Ito (2015). "The role of 5α-reductase type 1 associated with intratumoral dihydrotestosterone concentrations in human endometrial carcinoma." Mol Cell Endocrinol 401: 56-64. Tangen, I. L., T. B. Onyango, R. Kopperud, A. Berg, M. K. Halle, A. M. Øyan, H. M. Werner, J. Trovik, K. H. Kalland, H. B. Salvesen and C. Krakstad (2016). "Androgen receptor as potential therapeutic target in metastatic endometrial cancer." Oncotarget 7(31): 4928949298.

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Taylor, A. H., M. Guzail, M. Wahab, J. R. Thompson and F. Al-Azzawi (2005). "Quantitative histomorphometric analysis of gonadal steroid receptor distribution in the normal human endometrium through the menstrual cycle." Histochem Cell Biol 123(4-5): 463-474. Toraldo, G., S. Bhasin, M. Bakhit, W. Guo, C. Serra, J. D. Safer, J. Bhawan and R. Jasuja (2012). "Topical androgen antagonism promotes cutaneous wound healing without systemic androgen deprivation by blocking beta-catenin nuclear translocation and cross-talk with TGF-beta signaling in keratinocytes." Wound Repair Regen 20(1): 61-73. Tsai, S. J., M. H. Wu, C. C. Lin, H. S. Sun and H. M. Chen (2001). "Regulation of steroidogenic acute regulatory protein expression and progesterone production in endometriotic stromal cells." J Clin Endocrinol Metab 86(12): 5765-5773. Tuckerman, E. M., M. A. Okon, T. Li and S. M. Laird (2000). "Do androgens have a direct effect on endometrial function? An in vitro study." Fertil Steril 74(4): 771-779. Urban, R. R., N. N. Teng and D. S. Kapp (2011). "Gynecologic malignancies in female-to-male transgender patients: the need of original gender surveillance." Am J Obstet Gynecol 204(5): e9-e12. Vienonen, A., S. Miettinen, M. Bläuer, P. M. Martikainen, E. Tomás, P. K. Heinonen and T. Ylikomi (2004). "Expression of nuclear receptors and cofactors in human endometrium and myometrium." J Soc Gynecol Investig 11(2): 104-112. Walters, K. A., C. M. Allan, M. Jimenez, P. R. Lim, R. A. Davey, J. D. Zajac, P. Illingworth and D. J. Handelsman (2007). "Female mice haploinsufficient for an inactivated androgen receptor (AR) exhibit age-dependent defects that resemble the AR null phenotype of dysfunctional late follicle development, ovulation, and fertility." Endocrinology 148(8): 3674-3684. Walters, K. A., K. J. McTavish, M. G. Seneviratne, M. Jimenez, A. C. McMahon, C. M. Allan, L. A. Salamonsen and D. J. Handelsman (2009). "Subfertile female androgen receptor knockout mice exhibit defects in neuroendocrine signaling, intraovarian function, and uterine development but not uterine function." Endocrinology 150(7): 3274-3282. Whitaker, L. H., A. A. Murray, R. Matthews, G. Shaw, A. R. Williams, P. T. Saunders and H. O. Critchley (2017). "Selective progesterone receptor modulator (SPRM) ulipristal acetate (UPA) and its effects on the human endometrium." Hum Reprod 32(3): 531-543. Xu, J., M. Li, L. Zhang, H. Xiong, L. Lai, M. Guo, T. Zong, D. Zhang, B. Yang, L. Wu, M. Tang and H. Kuang (2015). "Expression and regulation of androgen receptor in the mouse uterus during early pregnancy and decidualization." Mol Reprod Dev. Yang, H., K. Kang, C. Cheng, R. Mamillapalli and H. S. Taylor (2015). "Integrative Analysis Reveals Regulatory Programs in Endometriosis." Reprod Sci 22(9): 1060-1072. Yang, H. P., J. Gonzalez Bosquet, Q. Li, E. A. Platz, L. A. Brinton, M. E. Sherman, J. V. Lacey, M. M. Gaudet, L. A. Burdette, J. D. Figueroa, J. G. Ciampa, J. Lissowska, B. Peplonska, S. J. Chanock and M. Garcia-Closas (2010). "Common genetic variation in the sex hormone metabolic pathway and endometrial cancer risk: pathway-based evaluation of candidate genes." Carcinogenesis 31(5): 827-833. Yarrow, J. F., S. C. McCoy and S. E. Borst (2012). "Intracrine and myotrophic roles of 5αreductase and androgens: a review." Med Sci Sports Exerc 44(5): 818-826. Yeh, S., M. Y. Tsai, Q. Xu, X. M. Mu, H. Lardy, K. E. Huang, H. Lin, S. D. Yeh, S. Altuwaijri, X. Zhou, L. Xing, B. F. Boyce, M. C. Hung, S. Zhang, L. Gan and C. Chang (2002). "Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues." Proc Natl Acad Sci U S A 99(21): 1349813503. Youngblood, G. L. and A. H. Payne (1992). "Isolation and characterization of the mouse P450 17 alpha-hydroxylase/C17-20-lyase gene (Cyp17): transcriptional regulation of the

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gene by cyclic adenosine 3',5'-monophosphate in MA-10 Leydig cells." Mol Endocrinol 6(6): 927-934. Zang, H., L. Sahlin, B. Masironi and A. L. Hirschberg (2008). "Effects of testosterone and estrogen treatment on the distribution of sex hormone receptors in the endometrium of postmenopausal women." Menopause 15(2): 233-239. Zhang, X. and B. A. Croy (1996). "Maintenance of decidual cell reaction by androgens in the mouse." Biol Reprod 55(3): 519-524.

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Figure legends Table 1: Expression of steroidogenic enzymes in the endometrium. Figure 1. Spatio-temporal changes in the endometrium during the human menstrual cycle. In the proliferative phase, ovarian-derived E2 induces endometrial growth. Current

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evidence suggests that, following ERα signalling, stromal-secreted IGF1 can bind on IGF1 receptors on epithelial cells, inducing proliferation. Following a mid-cycle peak in androgens, circulating progesterone concentrations gradually increase during the secretory phase and the

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endometrium undergoes decidualisation to provide a receptive environment for a prospective pregnancy. Expression of AR significantly decreases in stromal cells from the proliferative to the

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secretory phase. Stromal fibroblasts differentiate into secretory epithelioid-like decidual cells that begin to express and secrete the decidualisation-associated markers IGFBP1 and PRL, concomitant with expression of the steroidogenic enzymes AKR1C3 and CYP19A1. Testosterone and E2 synthesis from local steroid precursors can have intracrine and paracrine effects that impact on successful decidualisation.

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Figure 2. AR is present in nuclei of stromal cells but not detected in epithelial and endothelial cells in the mouse uterus. Uterine sections of female C57BL/6 mice were stained

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by dual immunofluorescence for the endothelial cell marker CD31 (in red) and AR (in green). Nuclear staining for AR was detected in stromal cells, while epithelial and endothelial (CD31positive) cells did not express AR. In the bottom panel, arrows indicate the location of individual

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endothelial cells in the different wavelength channels. DAPI (blue) was used as a nuclear counterstain. G=gland, St=stroma, L=lumen. Figure 3. Expression of MMP3 in mouse endometrium reveals DHT-dependent changes in in a mouse model of endometrial wound healing. Artificial decidualisation followed by

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progesterone withdrawal induced endometrial breakdown in female mice. Treatment with DHT resulted in temporal alterations in decidual shedding and re-epithelialisation and was accompanied by differences in MMP3 immunostaining patterns between untreated and treated

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groups. Scale bars: 200µm. NPC: no primary antibody control, LE: luminal epithelium, SC: shed cells (adapted from (Cousins, Kirkwood et al. 2016)).

1080 Figure

4.

Representative

endometrial

haematoxylin

and

eosin

stained

and

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immunohistochemistry-stained sections from cycling women and an individual undergoing long-term T treatment. Long-term androgen administration in a female-to-male (FtM) transitioning individual resulted in an atrophic endometrium of low thickness consisting of

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simple non-tortuous glands. AR is detected predominantly in the nuclei of stromal fibroblasts

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with few glandular epithelial cells exhibiting positive AR staining. Minimal proliferation was detected by Ki67 staining suggestive of an inactive state of the endometrium. Scale bars: H&E:

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1000 µm. AR/Ki67: 100 µm.

Figure 5. Androgens impact on the proliferation and decidualisation of endometrial cells. In the human endometrium, androgens have anti-proliferative effects both on epithelial and decidualising stromal cells. In addition, androgens upregulate the expression of decidualisation

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markers, with possible implications for implantation and pregnancy outcomes.

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+

+

Detection

mRNA

Reference

(Tsai, Wu et al. 2001, Aghajanova, Hamilton et al. 2009)

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Epithelium

Stroma

Enzyme

Whole-tissue

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(Hukkanen, Mantyla et al. 1998, Tsai, Wu et CYP11A1 +

+

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2009,

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2013, Huhtinen, Saloniemi-Heinonen et al.

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Hamilton et al. 2009)

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et

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Hamilton et al. 2009)

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protein

et al. 2006, Gibson, Simitsidellis et al. 2016)

mRNA, protein

(Bukulmez, Hardy et al. 2008, Aghajanova, Hamilton et al. 2009, Gibson, McInnes et al. 2013)

mRNA,

(Ito, Suzuki et al. 2002, Carneiro, Morsch et

protein

al. 2008, Gibson, Simitsidellis et al. 2016)

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ACCEPTED MANUSCRIPT Highlights •

Androgens control key functional processes that regulate endometrial function.

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Androgen receptors are most abundant in stromal cells of normal endometrium and in epithelial cells of endometrial cancers. New evidence suggests both local activation and metabolism of androgens are essential

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for endometrial competence during the establishment of pregnancy.

Dysregulation of androgen biosynthesis is associated with endometrial pathologies and impaired endometrial function.

Androgen receptors have important therapeutic potential for the regulation of

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endometrial function in health and disease.

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