**3. Abnormal folliculogenesis and impaired oocyte function**

Infertility associated with the advanced stages of endometriosis may be explained by pelvic adhesion and endometrioma as described above. The mechanism of infertility associated with endometriosis without adhesion and endometrioma, such as minimal or mild endometriosis as well as the negative impact of all stages of the disease on infertility is poorly understood. Many possibilities have been suggested, ranging from abnormal folliculogenesis to impaired endometrium receptivity (Arici *et al*.*,* 1999). Peritoneal fluid, a biologic fluid present in the abdominal cavity, has been a focus of research on endometriosis because of the extent of information it potentially carries about the disease. The proximity of peritoneal fluid to endometriotic lesions shows the milieu in which the immune mediators associated with the local inflammation of endometriosis can be studied. It has been suggested that such alterations in cytokines and growth factors interfere with folliculogenesis, ovulation and fertilization (Arici *et al*.*,* 1999).

The local microenvironment of peritoneal fluid surrounding the endometriotic implant is immunologically dynamic and links the reproductive and immune systems. Peritoneal fluid contains a variety of free floating cells, including macrophages, mesothelial cells, lymphocytes, eosinophils and mast cells (Oral *et al.,* 1996). The peritoneal fluid of women with endometriosis have confirmed an increased number, concentration and activation of macrophages which may induce proliferation of cells that are involved in inflammation through secretion of factors such as IL- 1, IL-6, and TNF-α (Oral *et al*., 1996a). Other studies similary found that levels of cytokines, such as, IL-6, IL-8 and TNF-α increased in the peritoneal fluid of women with endometriosis (Arici *et al*., 1996), meanwhile endometriotic implants also secreted various cytokines including IL-1, IL-6, IL-8, TNF-α in the peritoneal cavity in patients with endometriosis (Oral *et al*., 1996b). Cytokines, which are produced by many cell types in peritoneal fluid, play a diverse role as toxic effect in constructing the peritoneal environment that induces the development and progression of endometriosis and endometriosis-associated infertility (Harada *et al*., 2001).

Peritoneal fluid bathed the ovaries, hypothetically the inflammatory components in peritoneal fluid in women with endometriosis might diffuse into the ovarian follicles, or by

Pathomechanism of Infertility in Endometriosis 347

abnormal development. Alterations of the spindle may be one of the many causes related to infertility and/or recurrent pregnancy loss in patients with endometriosis (Mansour *et al*., 2010). Reactive oxygen species (ROS) have been detected in peritoeal fluid of endometriosis patients but are not significantly elevated compared with the control and idiopathic infertility groups (Bedaiwy *et al*., 2002). Reactive oxygen species have detrimental effects on oocytes, they are able to diffuse and pass through cell membrane and alter most types of cellular molecule such as lipids, proteins and nucleic acids. The consequences are mitochondrial alterations, embryo cell block, ATP depletion and apoptosis (Guerin, 2001). Based on several studies above it is proposed that pro-inflammatory factors and ROS in follicular fluid women with endometriosis may diffuse and impact autocrine-paracrine communication of ovarian follicles causing cell-cycle alteration and an increased apoptosis in granulosa cells. Beside that, the presence of pro-inflammatory factors and ROS could influence the oocyte such as abnormal meiotic spindle, chromosomal misalignment and decreased GDF-9 production. Both may impair oocyte-granulosa cell communicatin and cause abnormal folliculogenesis and, in turn, result in reduced oocyte quality. Futher studies

are needed. (see figure 1)

**4. Altered sperm function** 

Fig. 1. Abnormal folliculogenesis in endometriosis (Hendarto, 2011)

Spermatozoa have to stay for a certain period of time in the female genital tract that normally favors capacitation, the ability to reach and fertilize the oocyte. The endometriosis-associated immuno-inflammatory changes in peritoneal fluid may have some adverse effects on spermatozoa (Carli *et al.,* 2007). Eisermann (1989) reported that levels of TNF-α of up to 800 U/ml in peritoneal fluid from infertile women with endometriosis higher than fertile women without endometriosis. In this concentration,

paracrine mechanisms (Carlberg *et al*., 2000) impair the granulosa cell function, oocyte maturation and folliculogenesis. Folliculogenesis is growth and development process of ovarian follicle consist of oocyte, granulosa and theca cells might result in mature and fertilizable oocyte (Rajkovic, 2006). The alteration of oocyte, granulosa, theca cells development and molecular follicular communication may impact on folliculogenesis. Carlberg (2000) found that granulosa cells of women with endometriosis have an upregulated production of IL-1β, IL-6, IL-8, TNF-α which might be related to the reduced fertilization rate previously observed in endometriosis women (Carlberg *et al*., 2000). Beside that women with endometriosis were reported having higher granulosa cell apoptosis rate and a lower percentage of G2/M phase granulosa cells compared with other group of infertile women. This result strongly suggest that the cytokines produced in endometriosis women may be responsible for the disturbance of the cell cycle in the granulosa cells as in other cells and in turn have pathogenic effects on folliculogenesis (Toya *et al*. 2000) . Nakahara (1998) found that higher incidence of apoptotic bodies correlates with a lower quality of oocytes in individual follicles. This study showed that the incidence of apoptotic bodies in membrana granulosa ovaries of patients with endometriosis undergoing the IVF-ET procedure was increased as the stage of the revised AFS classification advanced. It means that the quality of oocytes from patients with endometriosis decreases in proportion to advancing stages of the revised AFS classification and determine the degree of disturbance for folliculogenesis in the ovaries of the patients with endometriosis (Nakahara, 1998).

Our previous study postulated that apoptosis of granulosa cells caused disturbance in oocyte growth and maturation and associated with decreased growth differentiation factor-9 (GDF-9) production (Hendarto *et al*., 2010). Oocyte-derived GDF-9 is obligatory for normal folliculogenesis and female fertility (Erickson and Shimasaki, 2001). Elvin (1999) reported that mouse GDF-9 can bind to receptors on granulosa cells, and plays multifunctional roles in oocyte-granulosa cell communication and regulation of follicular differentiation and function (Elvin *et al.*, 1999). In our study we found that the presence of GDF-9 in follicular fluid of preovulatory follicle was confirmed by western blotting analysis in a band of 53 kDa, and compared with the level in women with no endometriosis, GDF-9 level in the follicular fluid of women with severe endometriosis was lower. This might impair folliculogenesis, leading to reduced oocyte quality (Hendarto *et al*., 2010). Our other study also comfirmed that oocyte-granulosa cell communication has already been altered showed by increasing the concentration of granulosa cell-derived kit-ligand in follicular fluid of infertile women with endometriosis ( in publication process).

The cytoskeleton of metaphase II oocytes were influenced by rich pro-inflammatory factor present in peritoneal fluid of patients with endometriosis. By exposure of cryopreserved mouse oocytes to the peritoneal fluid from women with endometriosis, Mansour (2010) reported that in the endometriosis group, the cytoskeleton had a higher frequency of abnormal meiotic spindle and chromosomal misalignment, indicating severe damage compared with the control groups. The meiotic spindle plays a critical role in maintaining chromosomal organization and formation of the second polar body. Disorganization of the meiotic spindle can result in chromosomal dispersion, failure of normal fertilization, and

paracrine mechanisms (Carlberg *et al*., 2000) impair the granulosa cell function, oocyte maturation and folliculogenesis. Folliculogenesis is growth and development process of ovarian follicle consist of oocyte, granulosa and theca cells might result in mature and fertilizable oocyte (Rajkovic, 2006). The alteration of oocyte, granulosa, theca cells development and molecular follicular communication may impact on folliculogenesis. Carlberg (2000) found that granulosa cells of women with endometriosis have an upregulated production of IL-1β, IL-6, IL-8, TNF-α which might be related to the reduced fertilization rate previously observed in endometriosis women (Carlberg *et al*., 2000). Beside that women with endometriosis were reported having higher granulosa cell apoptosis rate and a lower percentage of G2/M phase granulosa cells compared with other group of infertile women. This result strongly suggest that the cytokines produced in endometriosis women may be responsible for the disturbance of the cell cycle in the granulosa cells as in other cells and in turn have pathogenic effects on folliculogenesis (Toya *et al*. 2000) . Nakahara (1998) found that higher incidence of apoptotic bodies correlates with a lower quality of oocytes in individual follicles. This study showed that the incidence of apoptotic bodies in membrana granulosa ovaries of patients with endometriosis undergoing the IVF-ET procedure was increased as the stage of the revised AFS classification advanced. It means that the quality of oocytes from patients with endometriosis decreases in proportion to advancing stages of the revised AFS classification and determine the degree of disturbance for folliculogenesis in the ovaries of the patients with endometriosis (Nakahara,

Our previous study postulated that apoptosis of granulosa cells caused disturbance in oocyte growth and maturation and associated with decreased growth differentiation factor-9 (GDF-9) production (Hendarto *et al*., 2010). Oocyte-derived GDF-9 is obligatory for normal folliculogenesis and female fertility (Erickson and Shimasaki, 2001). Elvin (1999) reported that mouse GDF-9 can bind to receptors on granulosa cells, and plays multifunctional roles in oocyte-granulosa cell communication and regulation of follicular differentiation and function (Elvin *et al.*, 1999). In our study we found that the presence of GDF-9 in follicular fluid of preovulatory follicle was confirmed by western blotting analysis in a band of 53 kDa, and compared with the level in women with no endometriosis, GDF-9 level in the follicular fluid of women with severe endometriosis was lower. This might impair folliculogenesis, leading to reduced oocyte quality (Hendarto *et al*., 2010). Our other study also comfirmed that oocyte-granulosa cell communication has already been altered showed by increasing the concentration of granulosa cell-derived kit-ligand in follicular fluid of infertile women with endometriosis

The cytoskeleton of metaphase II oocytes were influenced by rich pro-inflammatory factor present in peritoneal fluid of patients with endometriosis. By exposure of cryopreserved mouse oocytes to the peritoneal fluid from women with endometriosis, Mansour (2010) reported that in the endometriosis group, the cytoskeleton had a higher frequency of abnormal meiotic spindle and chromosomal misalignment, indicating severe damage compared with the control groups. The meiotic spindle plays a critical role in maintaining chromosomal organization and formation of the second polar body. Disorganization of the meiotic spindle can result in chromosomal dispersion, failure of normal fertilization, and

1998).

( in publication process).

abnormal development. Alterations of the spindle may be one of the many causes related to infertility and/or recurrent pregnancy loss in patients with endometriosis (Mansour *et al*., 2010). Reactive oxygen species (ROS) have been detected in peritoeal fluid of endometriosis patients but are not significantly elevated compared with the control and idiopathic infertility groups (Bedaiwy *et al*., 2002). Reactive oxygen species have detrimental effects on oocytes, they are able to diffuse and pass through cell membrane and alter most types of cellular molecule such as lipids, proteins and nucleic acids. The consequences are mitochondrial alterations, embryo cell block, ATP depletion and apoptosis (Guerin, 2001).

Based on several studies above it is proposed that pro-inflammatory factors and ROS in follicular fluid women with endometriosis may diffuse and impact autocrine-paracrine communication of ovarian follicles causing cell-cycle alteration and an increased apoptosis in granulosa cells. Beside that, the presence of pro-inflammatory factors and ROS could influence the oocyte such as abnormal meiotic spindle, chromosomal misalignment and decreased GDF-9 production. Both may impair oocyte-granulosa cell communicatin and cause abnormal folliculogenesis and, in turn, result in reduced oocyte quality. Futher studies are needed. (see figure 1)

Fig. 1. Abnormal folliculogenesis in endometriosis (Hendarto, 2011)

#### **4. Altered sperm function**

Spermatozoa have to stay for a certain period of time in the female genital tract that normally favors capacitation, the ability to reach and fertilize the oocyte. The endometriosis-associated immuno-inflammatory changes in peritoneal fluid may have some adverse effects on spermatozoa (Carli *et al.,* 2007). Eisermann (1989) reported that levels of TNF-α of up to 800 U/ml in peritoneal fluid from infertile women with endometriosis higher than fertile women without endometriosis. In this concentration,

Pathomechanism of Infertility in Endometriosis 349

showed a significantly reduced ability to implant compared with the remaining groups (Simon et al., 1994). These result above suggest that infertility in endometriosis patients may be related to alterations within the oocyte which, in turn, result in reduced embryos

Endometriosis induces an inflammatory state by activation of macrophages, releasing ROS and cytokines (Gupta *et al.,* 2008). Macrophages, cytokines and other products present in the peritoneal fluid from patients with endometriosis could be responsible for a change in the peritoneal environment that generates embryotoxic activity. Torres (2002) found embryotoxicity was increased in women with endometriosis, but there was little correlation with severity of the disease. These study also found a significant increase in embryotoxicity in the presence of high cytokine concentrations, especially with IL-6 (Torres et al., 2002). Other study by Pellicer found progesterone concentrations in follicular fluid increased with the severity of endometriosis that may be related to the release of the cytokines. The result of the study also showed that IL-6 concentration was significantly increased in follicular fluid of patients with endometriosis, whereas VEGF accumulation in follicular fluid was significantly decreased in women with endometriosis compared with controls. The increased IL-6 means that the immune system may be activated as a marker of altered follicular function that result in reduced oocyte and embryo quality. The decreased VEGF concentration needs further investigation, but in IVF, elevated VEGF concentrations have been shown to be related to good follicular vascularization and health. The study by Pellicer concluded profound differences in the follicular environment of the oocytes of women with endometriosis, compared with those of healthy patients. It may be suspected as a marker of altered follicular function that result in reduced oocyte and embryo quality.

There are controversial information regarding implantation alteration in endometriosisassociated infertility. Various studies described three causative factors: an oocyte/embryo impairment, endometrial defect and altered endometrial-embryonic cross-talk (Garrido *et al*., 2002). Implantation depends on an interaction of the trophoblast with the uterine epithelium, whereas a receptive endometrium is characterized by abundant secretory activity such as the presence of several integrins including the αvβ3 integrin. Lessey (1994) reported that the majority of women with abnormal αvβ3 integrin expression had endometriosis stage I or II and stated that αvβ3 integrin expression could be a useful marker of mild endometriosis (Lessey *et al.,* 1994). Inconsistent result pointed by Surrey (2010) that a high prevalence of aberrant endometrial αvβ3 vitronectin expression was noted in a group of infertile endometriosis patients who are IVF candidates but there were no significant differences in ongoing pregnancy or implantation rates in those patients who failed to express integrin αvβ3 vitronectin who were treated with a 3-month course of a GnRH agonist before an IVF cycle in comparison to untreated controls. Endometrial αvβ3 integrin expression did not predict which patients would benefit from prolonged administration of a GnRH agonist before initiation of controlled ovarian hyperstimulation for IVF (Surrey *et al.,*

The detection of pinopodes as a possible marker of receptivity in humans has been extensively studied. Pinopodes are specialized cell surface formations presumably involved

quality (Pellicer et al., 1995).

**6. Impaired endometrium receptivity** 

2010)

TNF-α caused a significant reduction in both progressive and total sperm motility when compared with controls group. Suggests that this may be a mechanism for the infertility observed in women with minimal endometriosis (Eisermann *et al*., 1989). In the other study stated that the toxic effects of TNF-α could be the result of its ability to stimulate apoptosis in sperm cells through initiation of a caspase cascade. Exposing spermatozoa to pathological concentrations of TNF-α can result in significant loss of sperm function and genomic integrity. Infliximab, an TNF-α inhibitor, could potentially be used to help treat female infertility caused by endometriosis in those with elevated levels of TNF-α in their peritoneal fluid (Said *et al.,* 2005).

Another theory describing pathological effect of endometriosis on sperm function is the role of reactive oxygen species. Oxidative stress has been shown to exert toxic effects on sperm, damaging the sperm cell membrane, inducing DNA damage, and mediating sperm apoptosis (Agarwal *et al.,* 2006). Mansour (2009) found that progressive sperm DNA damage was significantly higher in samples incubated with peritoneal fluid from patients with endometriosis than those from healthy women. Spermatozoa are particularly susceptible to ROS-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids and their cytoplasm contains low concentrations of the scavenging enzymes (Saleh *et al.,* 2002)

Reeve (2005) reported that significantly more spermatozoa bound per unit area to the ampullary epithelium of the uterine tubes taken from women with endometriosis, could potentially hinder fertilization by reducing the number of free spermatozoa in the tubal lumen that are available to take part in fertilization. Numerous studies have shown that spermatozoa that bind to the endosalpinx retain their viability, motility and fertilizing capacity longer than spermatozoa incubated alone or with other cell types. The aberrant expression of integrin in the endometrium of women with endometriosis would be speculated to increased sperm binding (Reeve *et al*., 2005).
