**2.1 Matrix turnover in endometrium and implantation**

The unique characteristic of the endometrial tissue is that it undergoes cyclic degeneration and regeneration in each menstrual cycle. The endometrium consists of a layer of columnar epithelium bedded on a layer of connective tissue. The extracellular matrix (ECM), which forms a component of the connective tissue, provides the scaffolding for the anchorage of the cells within the tissue (McIntush and Smith 1998) and presents a locale for cellular migration, division and differentiation (Birkedal-Hansen et al., 1993). The extensive remodeling of the connective tissue of the endometrium requires both the degradation and reformation of the ECM which is accomplished by highly regulated turnover of the ECM (Hulboy et al., 1997). This destruction of the ECM occurs by the action of a class of proteolytic enzyme identified as matrix metalloproteinases (MMP). MMPs and the tissue inhibitors of metalloproteinases (TIMP) regulate a number of aspects of reproductive physiology like dynamic remodeling of the ovary and endometrium throughout each menstrual cycle, implantation, embryonic development and parturition. There exists equilibrium between the MMPs and TIMPs action for controlling this turnover of the ECM. Any circumstances that may bring about interruption of this delicate balance leads to a number of pathological complexities related to pregnancy and infertility like luteinized unruptured follicle syndrome, ovarian cysts, endometriosis, uterine fibroids, inappropriate implantation resulting in tubal pregnancy or spontaneous abortion, premature rupture of fetal membranes, or carcinoma of the ovary or uterus (Curry and Osteen 2003).

Several studies have reported the diverse pattern of MMPs expression in the endometrium throughout the menstrual cycle. Since breakdown of the endometrial lining occurs during menstruation, several MMPs are reported to be highly expressed during this phase. However, their expression gradually falls off during rest of the cycle. Nevertheless various MMPs are observed to express at various phases of the cycling endometrium. Expression of MMP-2 remains consistent throughout the whole cycle along with TIMP-1 and TIMP-2. Endometrial MMP-9 expression shows a cyclical change in its distribution between glandular and stromal cells. It expresses in the endometrium throughout the cycle, however, its expression increases during midsecretory phase particularly in the glandular cells (Hulboy et al., 1997). Although the association of MMP-2 and -9 and their endogenous inhibitors in pathogenesis of infertile condition like endometriosis is well established (Salata et al., 2008), knowledge regarding their involvement in endometrial remodelling during implantation window in endometriotic women is limited.

#### **2.2 Angiogenesis**

Endometrial remodeling involves proliferation of its functional layer upon estrogen enhancement (Groothuis et al., 2007) and differentiation by the influence of progesterone from the luteinized follicles (Okada et al., 1999). This is followed by the degeneration of this superficial layer and again reconstruction of the new one. These constant cyclic changes of the endometrium are associated with angiogenesis and neovascularisation (Perrot-Applanat et al., 2000). Vascular endothelial growth factor (VEGF) is a prime angiogenic stimulus for vascular permeability based on its capability to bring on vascular leakage (Ferrara and Davis-Smyth 1997, Ferrara et al., 2003). VEGF regulated angiogenesis and neovasculogenesis of the endometrial tissue is elemental for the growth and differentiation of the endometrium for implantation and placentation (Giudice 1996, Perrot-Applanat 2000). Due to its control over the human reproductive cycle, VEGF is present in the stromal and glandular epithelium of the human endometrium throughout all phases of the menstrual cycle (Torry and Torry 1997, Smith 1998, Shifren et al., 1996, Charnock-Jones 1993, Popovici 1999, Lockwood 2002). However there exists a strong debate regarding its expression and angiogenesis. VEGF expression increased in the late secretory phase and heightened during menses (Torry and Torry 1997, Charnock-Jones 1993, Popovici 1999, Lockwood 2002, Bausero 1998). There is a marked increase even in the vascular network of the endometrium during the secretory phase over the proliferative phase (Ota 1998). But Nayak and Brenner reported that during proliferative phase there is a noted increase in the VEGF expression in stroma which shifts to glandular epithelium during the secretory phase (Nayak and Brenner 2002). However, contradictory report exist indicating that a gradual decline in angiogenesis occurs at the end of the cycle which rapidly increases with the start of a new cycle and reaching a maximum height during the mid cycle (Au and Rogers 1993). Other investigators have suggested that VEGF expression remains inconsistent (Sugino et al., 2002) or there is no change in the vascularity throughout the endometrial cycle (Rogers and Au 1993).

#### **2.3 Regulation of matrix remodeling**

326 Endometriosis - Basic Concepts and Current Research Trends

The unique characteristic of the endometrial tissue is that it undergoes cyclic degeneration and regeneration in each menstrual cycle. The endometrium consists of a layer of columnar epithelium bedded on a layer of connective tissue. The extracellular matrix (ECM), which forms a component of the connective tissue, provides the scaffolding for the anchorage of the cells within the tissue (McIntush and Smith 1998) and presents a locale for cellular migration, division and differentiation (Birkedal-Hansen et al., 1993). The extensive remodeling of the connective tissue of the endometrium requires both the degradation and reformation of the ECM which is accomplished by highly regulated turnover of the ECM (Hulboy et al., 1997). This destruction of the ECM occurs by the action of a class of proteolytic enzyme identified as matrix metalloproteinases (MMP). MMPs and the tissue inhibitors of metalloproteinases (TIMP) regulate a number of aspects of reproductive physiology like dynamic remodeling of the ovary and endometrium throughout each menstrual cycle, implantation, embryonic development and parturition. There exists equilibrium between the MMPs and TIMPs action for controlling this turnover of the ECM. Any circumstances that may bring about interruption of this delicate balance leads to a number of pathological complexities related to pregnancy and infertility like luteinized unruptured follicle syndrome, ovarian cysts, endometriosis, uterine fibroids, inappropriate implantation resulting in tubal pregnancy or spontaneous abortion, premature rupture of

fetal membranes, or carcinoma of the ovary or uterus (Curry and Osteen 2003).

implantation window in endometriotic women is limited.

**2.2 Angiogenesis** 

Several studies have reported the diverse pattern of MMPs expression in the endometrium throughout the menstrual cycle. Since breakdown of the endometrial lining occurs during menstruation, several MMPs are reported to be highly expressed during this phase. However, their expression gradually falls off during rest of the cycle. Nevertheless various MMPs are observed to express at various phases of the cycling endometrium. Expression of MMP-2 remains consistent throughout the whole cycle along with TIMP-1 and TIMP-2. Endometrial MMP-9 expression shows a cyclical change in its distribution between glandular and stromal cells. It expresses in the endometrium throughout the cycle, however, its expression increases during midsecretory phase particularly in the glandular cells (Hulboy et al., 1997). Although the association of MMP-2 and -9 and their endogenous inhibitors in pathogenesis of infertile condition like endometriosis is well established (Salata et al., 2008), knowledge regarding their involvement in endometrial remodelling during

Endometrial remodeling involves proliferation of its functional layer upon estrogen enhancement (Groothuis et al., 2007) and differentiation by the influence of progesterone from the luteinized follicles (Okada et al., 1999). This is followed by the degeneration of this superficial layer and again reconstruction of the new one. These constant cyclic changes of the endometrium are associated with angiogenesis and neovascularisation (Perrot-Applanat et al., 2000). Vascular endothelial growth factor (VEGF) is a prime angiogenic stimulus for vascular permeability based on its capability to bring on vascular leakage (Ferrara and Davis-Smyth 1997, Ferrara et al., 2003). VEGF regulated angiogenesis and neovasculogenesis of the endometrial tissue is elemental for the growth and differentiation of the endometrium for implantation and placentation (Giudice 1996, Perrot-Applanat 2000). Due to its control

**2. Matrix turnover and angiogenesis during implantation** 

**2.1 Matrix turnover in endometrium and implantation** 

It is suggested that inappropriate regulation of sex steroids may lead to defect in implantation. The role of estradiol in embryo implantation is a subject of controversy and its association with pregnancy outcome in IVF cycle is an area of research for many years (Kyrou et al 2009). Several studies have shown that midluteal decline of serum estradiol do not affect the endometrial development, embryo implantation and IVF outcome (Friedler et al., 2005; Narvekar et al., 2010; Hung et al., 2000). This may be due to the fact that during follicular phase, estradiol induces growth of follicles, preparation of endometrium and production of specific proteins, growth factors and receptors of estrogen and progesterone. Additionally, adverse effect of high estradiol level on endometrial receptivity is still under debate (Kyrou et al., 2009). A number of investigators found no effect of high estradiol levels on the treatment outcome of IVF/ICSI cycle (Sharara and McClamrock 1999, Kosmas et al., 2004). Some studies have, however, suggested that elevated levels of estradiol may be responsible for impaired endometrial receptivity (Simon et al., 1995; Valbuena et al., 2001; Kyrou et al., 2009). After ovulation, progesterone is the main contributory sex hormone executing the transformation of the endometrium during the secretory phase.

It is evidenced that expression of cyclooxygenase-2 (COX-2), a molecule associated with angiogenesis and cell differentiation, promotes the release of MMP-2 (Xiong et al., 2005) and -9 (Itatsu et al., 2009), and angiogenic factor VEGF (Wang *et al.* (2010). COX-2, on the other hand, is regulated by female sexual hormone estradiol and progesterone (Li et al., 2007). Since the process of angiogenesis during endometrial remodelling shares similarities with the process of angiogenesis during metastasis in cancer, estradiol may also be involved in the up-regulation of the gene expression of COX-2 and MMPs during embryo implantation. Involvement of COX-2 gene in embryo implantation is a subject of interest among the researchers working on endometrial receptivity, and is suggested to play an important regulatory role in successful implantation. However, little is known about its role in endometrial receptivity in women with endometriosis.

#### **2.4 Endometrial receptivity markers**

Inadequate uterine receptivity and poor embryo formation are two major factors responsible for implantation failure (Simon et al., 1998; Ledee-Bataille et al., 2002). Nowadays, using

Alteration in Endometrial Remodeling: A Cause for Implantation Failure in Endometriosis? 329

the 14 cloned human mucins, only Mucin-1 (MUC1) and to a lesser extent MUC6 have been found in the human endometrium (Gipson et al., 1997). Cell–cell and cell–matrix adhesion are inhibited in direct correlation to the length of the MUC-1 ectodomain (Hilkens et al.,

Selectins are glycoproteins which also belong to the CAM family. The expression of selectin oligosaccharide-based ligands, such as MECA-79 or HECA-452, is up-regulated during the window of implantation (Genbacev et al., 2003). MECA-79 is immunolocalized in the luminal and glandular endometrial epithelium throughout the menstrual cycle, although the staining considerably intensifies during the mid-secretory phase. The physiological importance of the interaction between L-selectin and its oligosaccharide ligands has been

Though several studies investigating endometrial receptivity during implantation window are documented, the mechanism responsible for implantation failure in endometriosis is still poorly understood. Expression of various cell adhesion molecules and pinopodes in women with endometriosis is explored in the present study. Since, COX-2 is reported to be physiologically involved in the process of angiogenesis (Matsumoto et al., 2002), and in view of the fact that angiogenesis is essential for endometrial remodeling, we were motivated to assess the expression of various angiogenic factors including VEGFR, MMP-2,-9 and their tissue inhibitors in women with endometriosis during the implantation window. Additionally, expression of COX-2 was studied to assess their associated regulatory role in the process of endometrial remodeling

30 women with endometriosis and 20 without the disease were included in the study. Presence/absence of endometriosis was confirmed by diagnostic laparoscopy. It was ensured that these women had not received any kind of medical or hormonal treatment during the past three months. Women with history of chocolate cyst removal, previous history of any surgery, with other possible causes of pain or pelvic pathology including

Blood samples collected from patients were allowed to clot and the serum separated by centrifugation at 3,000 rpm for 5 min at 4°C. Serum samples were stored at -20°C until further use. Endometrial biopsy was performed on the 7th day after confirmation of ovulation. The collected tissue was washed in phosphate buffer saline (PBS) and divided into three parts: one part was used for stromal and epithelial cells isolation for flow cytometric analysis of different molecular repertoires of the endometrium, the other part was fixed for immunohistochemistry (IHC) and scanning electron microscopy of these

receptivity markers. From the third part, RNA was isolated immediately.

investigated in the human endometrium (Genbacev et al., 2003).

1992; Wesseling et al., 1996).

during implantation window.

pelvic tuberculosis were excluded.

**2.5.2 Sample collection** 

**2.5 Material and method 2.5.1 Subject selection** 

**2.4.5 L-Selectin ligand** 

ART procedure, clinicians can improve embryo formation considerably; however, no therapies are available to make the endometrium more receptive. Expression of various implantation markers and proteins lead to remodeling of the endometrial matrix thereby transforming the endometrium towards a receptive milieu. Several molecular repertoires expressed during the implantation window are considered to be useful markers of implantation. Expression of various markers including pinopodes αvβ3 integrin, LIF, Lselectin ligand and Mucin-1 throughout the different stages of implantation are considered to be responsible for endometrial receptivity.

#### **2.4.1 Pinopodes**

Pinopodes, also known as uterodomes, are large cytoplasmic protrusions from the endometrial epithelial surface and are several micrometers wide. These are specialized cell structures that are involved in adhesion and penetration of the blastocyst into the stroma. These structures project into the uterine lumen and are above the microvilli level. Their expression is limited to a maximum period of 2 days during the menstrual cycle corresponding to the presumed window of implantation (Stavreus-Evers et al., 2001). Endometrial pinopodes development is associated with the mid-luteal phase increased expression of leukaemia inhibitory factor (LIF) and its receptor (Aghajanova et al., 2003), progesterone (Stavreus-Evers et al., 2001) and integrin αVβ3 (Lessey et al., 1992). Advocated as a marker of uterine receptivity, their expression, has been investigated solely by means of scanning electron microscopy (SEM) (Develioglu et al., 2000).

#### **2.4.2 Integrins**

Integrins are surface ligands, usually glycoproteins, belonging to the class of cell adhesion molecules (CAM). An integrin molecule consists of two different, non-covalently linked α and β subunits that are paired to form various heterodimers with distinct function (Hynes, 2002). At least 20 types of integrin heterodimer have been defined, which form from 14α and 9β subunits (Lindhard 2002). Integrins are unusual cell surface receptors in that they bind with low affinity and are present in large numbers, allowing for ligand motility without loss of attachment. Endometrial epithelial cells constitutively express certain integrins, whereas others are cycle dependent (Lessey 1992). αvβ3, an example of the latter is present on the apical surface of both luminal endometrial cells and human embryos. 41 different aberrant expressions of this integrin are reported in women with endometriosis (Lessey et al., 1994).

#### **2.4.3 LIF**

LIF is a member of the IL-6 family and is secreted by the endometrial epithelium, CD16– CD56 natural killer cells and type 2 T-helper cells. Animal and human studies indicate that LIF plays an important role in implantation and for pregnancy to occur (Lass et al., 2001). LIF protein can be detected by immunohistochemistry in the luminal, glandular and stromal epithelium. There is very little LIF expression in proliferative endometrium, but levels increase during the secretory phase, reaching a maximum between days 19 and 25, which coincides with the implantation window (Charnock-Jones 1994).

#### **2.4.4 Mucins**

Mucins are high molecular weight (MW) glycoproteins, which contain at least 50% of carbohydrate O-linked to a threonine/serine rich peptide core (Gendler et al., 1990). Among the 14 cloned human mucins, only Mucin-1 (MUC1) and to a lesser extent MUC6 have been found in the human endometrium (Gipson et al., 1997). Cell–cell and cell–matrix adhesion are inhibited in direct correlation to the length of the MUC-1 ectodomain (Hilkens et al., 1992; Wesseling et al., 1996).
