**2. Role of endometrial prostaglandins in reproduction**

The uterus plays a crucial role in the implantation process of the fertilized egg. Humans and old-world primates have the particularity that they shed part of their endometrium if fertilization or implantation does not take place. Consequently, endometrium is a dynamic tissue that undergoes regulated phases of proliferation, differentiation and degradation, cyclically. PGs are known to be important actors of this process; different reports have implicated COX enzymes and PGs in several stages of reproduction (Jabbour et al., 2006).

COX-2 is physiologically expressed at different levels along the menstrual cycle. It has been shown to be expressed at its lowest level during ovulation and slightly start augmenting during the secretory phase, peaking at the late secretory and menstrual phases after which it starts decreasing again (Jones et al., 1997).

The importance of COX and PGs in reproduction has been revealed from studies of knockout mice. Even if COX-1 deficient mice were fertile, their gestation period was prolonged while parturition and viable offspring were reduced. These findings demonstrated that COX-1 is essential for normal labor in the mouse (Gross et al., 1998; Jabbour et al., 2006). On the other side, selective COX-2 ablation impairs ovulation, fertilization and implantation; and a combined approach showed that COX-2 inhibition in COX-1(-/-) mice induced complete reproductive failure, suggesting a lack of alternative sources of PG synthesis (Reese et al., 2001).

synthase (PGES), PGF synthase (PGFS), PGI synthase (PGIS) and TX synthase (TXS), respectively. Once synthesized prostanoids are rapidly exported by a PG transporter out of the cell and they function very close to their liberation site, in an autocrine or paracrine fashion. They exert their biological actions through G protein coupled receptors (GPCRs) and, as it happens with the synthases, each prostanoid has a distinctive receptor to which to bind to. DP, EP, FP, IP and TP are the receptors for PGD2, PGE2, PGF2α, PGI2 and TXA2, respectively (Figure 1). The EP receptor has four known subtypes (EP1-EP4), each encoded by a different gene; furthermore, EP3 has eight splice variants; TP and FP have also been

Sequence homology analysis revealed that receptors sharing a common signaling pathway are more closely related than do receptors binding the same ligand. After binding to the corresponding GPCR there is generation of soluble second messengers. Coupled to Gq, DP receptor increases cyclic adenosine monophosphate (cAMP) concentration, whereas IP receptor is coupled to Gs and increases not only cAMP but also mediates responses by phosphatidylinositol increasing free Ca2+ concentration (Narumiya et al., 1999). Both isoforms of TP activate phopspholipase C (PLC), but TPα activates adenylate cyclase while TPβ inhibits it (Narumiya et al., 1999). FP receptors also act through Gq, PLC and Ca2+ release; while EP receptors have distinctive signaling pathways depending on the subtype binding PGE2: EP1 is coupled to Gi and Ca2+ channels, EP2 and EP4 share the pathway coupling to Gs and increasing cAMP intracellular concentration, whereas the EP3 has specific responses depending on the splice variant, but is usually assumed as an inhibitory

The uterus plays a crucial role in the implantation process of the fertilized egg. Humans and old-world primates have the particularity that they shed part of their endometrium if fertilization or implantation does not take place. Consequently, endometrium is a dynamic tissue that undergoes regulated phases of proliferation, differentiation and degradation, cyclically. PGs are known to be important actors of this process; different reports have implicated COX enzymes and PGs in several stages of reproduction (Jabbour

COX-2 is physiologically expressed at different levels along the menstrual cycle. It has been shown to be expressed at its lowest level during ovulation and slightly start augmenting during the secretory phase, peaking at the late secretory and menstrual phases after which it

The importance of COX and PGs in reproduction has been revealed from studies of knockout mice. Even if COX-1 deficient mice were fertile, their gestation period was prolonged while parturition and viable offspring were reduced. These findings demonstrated that COX-1 is essential for normal labor in the mouse (Gross et al., 1998; Jabbour et al., 2006). On the other side, selective COX-2 ablation impairs ovulation, fertilization and implantation; and a combined approach showed that COX-2 inhibition in COX-1(-/-) mice induced complete reproductive failure, suggesting a lack of alternative

described to have two splice variants each (Fortier et al., 2008).

receptor coupled to Gi (Fortier et al., 2008) (Figure 1).

starts decreasing again (Jones et al., 1997).

sources of PG synthesis (Reese et al., 2001).

et al., 2006).

**2. Role of endometrial prostaglandins in reproduction** 

Arachidonic acid is the precursor for leukotrienes and prostaglandins. Each prostaglandin has a specific seven transmembrane G protein coupled receptor; after binding with its receptor, prostaglandins produce the up (↓) or downregulation (↑) of second messengers.

**AA**: arachidonic acid; **LTs**: leukotrienes; **LOX**: lipooxygenase; **COX-1/2**: cyclooxygenase-1 or 2; **PGH2:**  prostaglandin H2; **PGIS**: prostacyclin synthase; **PGI2**: prostacyclin; **TXS**: thromboxane synthase; **TXA2**: thromboxane; **PGE2:** prostaglandin E2**; PGD2:** prostaglandin D2**; PGF2:** prostaglandin F2**; PGES**: PGE2 synthase; **PGDS**: PGD2 synthase; **PGFS**: PGF2α synthase; **IP, TPα/β, EP1-4, DP, FPα/β**: specific PG receptors; **cAMP**: cyclic adenosine monophosphate; **IP3**: inositol triphosphate; **Ca2+**: calcium.

Fig. 1. Prostaglandin synthesis and signal transduction

In addition, studies using EP and FP knockout mice have demonstrated the specific roles of PGE2 and PGF2α in reproduction. It has been shown that EP2 receptors are essential for ovulation and fertilization (Kennedy et al., 1999; Ushikubi et al., 2000) and FP are indispensible for parturition (Sugimoto et al., 1998). These studies indicate not only the essential role of PGE2 in the fertilization process, but also the importance of PGF2α in natural parturition.

As well, it has been described that PGs serve as endogenous ligands for nuclear receptors. In this respect, other prostanoids were identified as good peroxisome proliferator-activated receptors (PPAR) agonists with varying specificity. 15-deoxy-Δ 12,14 prostaglandin J2 (15dPGJ2), a natural PPARγ ligand, has high affinity for PPARγ and has been proposed as a regulator of the inflammatory response (Nosjean & Boutin, 2002; Scher & Pillinger, 2009). Another PPAR ligand is PGI2 that was found to play an important role via PPAR-δ nuclear receptor in implantation and decidualization (Pakrasi & Jain, 2008).

The process of implantation is considered to be analogous to pro-inflammatory responses, hence the speculation that PGs play a role in this event (Kennedy, 1979; Maybin et al., 2011; Tranguch et al., 2005). As well, several nonsteroidal anti-inflammatory drugs (NSAIDs) and

Involvement of Prostaglandins in the Pathophysiology of Endometriosis 119

The mitogenic effects of estrogens are mediated by the upregulation of several growth factors and also by PGs. Specifically, aromatase and steroidogenic acute regulatory protein (StAR) are known to be regulated by PGE2 in endometriotic stromal cells (Bulun et al., 2004; Noble et al., 1997; Sun et al., 2003; Tsai et al., 2001). PGE2 alone via the EP2/EP4 receptor is sufficient to induce de novo synthesis of estrogen from cholesterol (Attar et al., 2009). As well, estrogen further stimulates the synthesis of PGE2 in ectopic endometrial tissue (Bulun et al., 2000; Noble et al., 1997). In conclusion, estrogens, pro-inflammatory and proangiogenic peptides contribute to elevate the expression of COX-2 and consequently the levels of PGE2 in endometriotic tissue and in peritoneal macrophages from patients with

Aromatase is the key enzyme in the conversion of the androgens, androstenedione and testosterone, to estrone and estradiol (E2) respectively (Bulun et al., 2001). This protein was seen to be overexpressed in the eutopic endometrium of patients with endometriosis compared to controls (Noble et al., 1996) and it has been described to be expressed in the ectopic endometriotic lesion. PGE2 induces not only aromatase expression but also its activity, as seen in studies conducted in endometriotic stromal cells *in vitro* (Noble et al., 1997); and its product, E2, induces COX-2 expression with the consequent synthesis of PGE2 (Tamura et al., 2004). It is clear that given the way these molecules interact, a positive feedback loop is established favoring the activity of aromatase, provoking high levels of E2 locally in the vicinity of the lesion (Figure 2). These high levels of E2 also give the endometriotic cells a high capability of proliferating; as it has been demonstrated that, through its estrogen receptor (ER)β, E2 enhances stromal cell proliferation (Trukhacheva et

Another mitogenic factor is fibroblast growth factor (FGF)-9. This molecule was found to be regulated by estrogen in endometriotic stromal cells in culture and, if added exogenously, cell proliferation was enhanced in a dose dependent manner. On the contrast, when cells were incubated with an aromatase or an ER inhibitor, the rate of cell proliferation diminished significantly compared to the untreated control (Wing et al., 2003). In the same study by Wing and coworkers, it was observed that not only FGF-9 is regulated by E2, but also, FGF receptors 2IIIc and 3IIIc. More recently, a study revealed that PGE2, acting through its receptor EP3 induces the expression of FGF-9 in a dose dependent manner in

Endometrial cells at the ectopic site are urged to establish their own irrigation network, this is essential for the further maintenance and growth of the endometriotic lesion. It is widely known that VEGF is crucial for the process of angiogenesis; this is the process by which new blood vessels can be developed from preexisting ones. It has been shown that patients with endometriosis have a higher VEGF concentration in peritoneal fluid than endometriosis free women (Mahnke et al., 2000). Moreover, it was seen that VEGF stimulates COX-2 expression (Tamura et al., 2006) and that PGE2 increases VEGF production (Gately & Li, 2004; Liu et al.,

endometriotic cells *in vitro* (Chuang et al., 2006) (Figure 2).

**3.3 PGE2 and angiogenesis** 

**3.2 Regulation of aromatase activation and estrogen production by PGE2**

endometriosis (Figure 2).

al., 2009).

selective COX-2 inhibitors were implicated in the inhibition of endometrial vascular permeability and implantation in a variety of species (Diao et al., 2007; Sookvanichsilp & Pulbutr, 2002).

In particular in the endometrium, COXs and PGs are known to be involved in the initiation of implantation and decidualization (Kennedy et al., 2007; Tranguch et al., 2005). It is well known that endometrial vascular permeability and proliferation and differentiation of endometrial stromal cells to decidual cells are mediated by PGs (Kennedy, 1979; Kennedy & Doktorcik, 1988). The initial studies of Chakraborty and coworkers suggest an important role for PGs in the implantation process; specifically this report demonstrated that COX-2 expression during the blastocyst attachment is critical to implantation (Chakraborty et al., 1996).

In an effort to identify which PGs are involved in the implantation process, different researchers have confirmed the presence of PGE and PGF receptors in the peri-implantation endometrium (Kennedy et al., 2007). However, no single type of PG has been unequivocally associated to this event. There may be species differences and also different PGs may be involved in the implantation or decidualization processes (Kennedy et al., 2007).
