**7. Endometriosis, steroidogenesis and folliculogenesis**

Some studies have shown an increase of luteinized unruptured follicle syndrome (LUF) and of the incidence of lutheal phase defects in women with endometriosis (Cheesman et al., 1983; Holtz et al., 1985; Saracoglu et al., 1985; Kaya & Oral, 1999). Other recent studies have shown a polymorphism of the progesterone gene and resistance to the action of progesterone in endometriosis tissues (Bulun et al., 2006; Van Kaam et al., 2007), supporting the hypothesis of impaired progesterone production and/or action in endometriosis (Bulun et al., 2006; Harlow et al., 1996). Some data show impaired steroidogenesis of granulosa cells associated with minimal endometriosis, represented not only by a reduction of basal aromatase activity, but also by a lower production of progesterone in stimulated as well as non-stimulated cycles (Harlow et al, 1996). Therefore, ovulatory dysfunction induced by impairment of ovarian steroid secretion as well as inadequate lutheal function might be important for the pathogenesis of infertility associated with endometriosis. A function defect in the oocyte due to abnormal follicle function might be the result of this ovulatory dysfunction (Wardle et al., 1985). Supporting this hypothesis, clinical studies involving IVF and some programs of oocyte donation have pointed out the importance of impaired oocyte quality in the pathogenesis of infertility associated with endometriosis (Pellicer et al., 1998; Garrido et al., 2002).

also with the transformation of the sperm head to male pronucleus (De Matos & Furnus, 2000). However, no studies have evaluated the expression of this enzyme or of the entire GSH redox system in COCs of patients with infertility related to endometriosis. Matos et al. (2009) suggested a positive correlation between the SOD activity of COCs of infertile women submitted o ovarian stimulation for ART due to male factor and ART outcomes. In this same study an increase in SOD activity was observed in in vitro culture of COCs from infertile women with endometriosis. However, the authors analyzed the COCs of only six

Some authors have associated minimal endometriosis with impaired steroidogenesis in granulosa cells, represented not only by a reduced baseline activity of aromatase, but also by a lower production of progesterone in non-stimulated and stimulated cycles (Harlow et al., 1996; Gomes et al., 2008). A functional failure of oocytes due to abnormal follicular function could be a result of this disease (Wardle et al., 1985). The antioxidants not only have an antiapoptotic effect on preovulatory in vitro cultured follicles (Tsai-Turton & Luderer, 2006), but are also involved in the regulation of steroidogenic enzyme function dependent on cytochrome P450 (Verit et al., 2007). Some studies have suggested that ascorbic acid (Murray et al., 2001), as well as SOD (Lapolt & Hong, 1995) may have inhibitory effects on aromatase, an enzyme responsible for the conversion of androgens to estrogens, which could induce storage of androgens in the follicular fluid, leading to follicular atresia (Verit et al., 2007). As mentioned earlier, some recent data have demonstrated an increase of SOD activity in COCs (in vitro culture) of infertile women with endometriosis (Matos et al., 2009). Since no studies on endometriosis patients have evaluated antioxidant enzyme expression in luteinized granulosa cells and their correlation with steroidogenic enzymes dependent on cytochrome P450 expression, involved in ovarian steroidogenesis, our group has performed studies

Some studies have shown an increase of luteinized unruptured follicle syndrome (LUF) and of the incidence of lutheal phase defects in women with endometriosis (Cheesman et al., 1983; Holtz et al., 1985; Saracoglu et al., 1985; Kaya & Oral, 1999). Other recent studies have shown a polymorphism of the progesterone gene and resistance to the action of progesterone in endometriosis tissues (Bulun et al., 2006; Van Kaam et al., 2007), supporting the hypothesis of impaired progesterone production and/or action in endometriosis (Bulun et al., 2006; Harlow et al., 1996). Some data show impaired steroidogenesis of granulosa cells associated with minimal endometriosis, represented not only by a reduction of basal aromatase activity, but also by a lower production of progesterone in stimulated as well as non-stimulated cycles (Harlow et al, 1996). Therefore, ovulatory dysfunction induced by impairment of ovarian steroid secretion as well as inadequate lutheal function might be important for the pathogenesis of infertility associated with endometriosis. A function defect in the oocyte due to abnormal follicle function might be the result of this ovulatory dysfunction (Wardle et al., 1985). Supporting this hypothesis, clinical studies involving IVF and some programs of oocyte donation have pointed out the importance of impaired oocyte quality in the pathogenesis of infertility associated with endometriosis (Pellicer et al., 1998;

patients.

evaluating these possible associations.

Garrido et al., 2002).

**7. Endometriosis, steroidogenesis and folliculogenesis** 

3β-Hydroxysteroide dehydrogenase/delta 5-delta 4-isomerase (3β-HSD) is an important enzyme associated with the biosynthesis of progesterone. Bar Ami (1994) evaluated the fertilization capacity related to the competence of granulosa cells and COCs to secrete progesterone. COCs from fertilized oocytes presented a 1.9 times higher progesterone level (p<0.001) on days 0-3 and a 1.6 times higher level (p<0.02) on days 3-5 of culture when compared to the levels in COCs of non-fertilized oocytes. Nevertheless, in COCs of fertilized oocytes, the activity of 3β-hydroxysteroid dehydrogenase was significantly higher after oocyte aspiration and also 3 to 5 days later compared to non-fertilized oocytes. These results suggest that, in stimulated cycles, in follicles that hold mature COCs there is a synchrony and correlation between competence to perform progesterone secretion by COCs as well as by granulosa cells and the potential of these oocytes to be fertilized. Such correlation suggests and supports the intimate relation of enzymatic activity of 3β-hydroxysteroid dehydrogenase and progesterone production with oocyte fertilization capacity, which may suggest the important role of this enzyme as coadjuvant in the acquisition of oocyte competence. The reduction of the gene expression and/or activity of this enzyme could lead to a lower production of progesterone and impairment of the lutheal phase.

Aromatase is present in granulosa cells and actually plays a fundamental role in follicle maturation and in the establishment of oocyte quality (Erickson et al., 1989; Foldesi et al., 1998; Speroff & Fritz, 2005). But, if on the one hand we find evidence of increased aromatase expression in ectopic endometrium, on the other, there are poor and inconclusive data concerning the expression of this enzyme by luteinized granulosa cells, suggesting a lower activity of this enzyme, but with no confirmation of an associated lower gene expression.

It is known that oocyte quality results from a complex and synchronized process that lasts several months, from primordial follicle to pre-ovulatory follicle. This process starts in a gonadotropin-independent way and later becomes gonadotropin dependent. In this last phase, oocyte, granulosa cells and FSH interact synergically. Granulosa cell multiplication and the specific way they respond first to FSH and later to LH in order to produce intrafollicle steroids are crucial events in this process (Speroff & Fritz, 2005). We know that there are gap junctions between granulosa cells, which is evidence that there are molecular interactions between them and possibly with the oocyte itself, through signaling molecules such as growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP 15) (Albertini & Barrett, 2003; Combelles et al., 2004; Thomas & Vanderhyden, 2006; Hutt & Albertini, 2007). However, little information is available about the communication between granulosa cells and the oocyte.

Granulosa cells differentiate into mural and cumulus cells during folliculogenesis, a fact that has stimulated the study of their potential as mesenchymal stem cells. To date there are no studies comparing the gene expression of mural granulosa cells and COCs and, possibly, since they are cells with distinct function and differentiation, there might be genes with different patterns of expression. When they reach the pre-antral follicle stage, granulosa cells can synthetize all three types of steroids (androgens, progestagens and estrogens) (Speroff & Fritz, 2005). However, the proportions and timing of their production are crucial. It is known that FSH and also LH have hormonal receptors on granulosa cells and there is a synergism between these receptors and intra-follicle hormonal production to permit the

Endometriosis and Infertility: The Role of Oxidative Stress 409

ART. However, data obtained by the analysis of gene expression of mural granulosa cells of patients with endometriosis cannot be necessarily extrapolated to cumulus oophorus cells

We found evidence that COCs might contribute to oocyte cytoplasmic maturation (Tanghe et al., 2002) through a net of gap junctions between COCs and between these and the oocyte (Furger et al., 1996). Nevertheless, the presence of COCs is important for fertilization to occur (Tanghe et al., 2002) because it attracts selected spermatozoa and promotes their capacitation and penetration. On the other hand, it should be emphasized that COCs protect the oocyte against apoptosis induced by oxidative stress (Tatemoto et al., 2000), which occurs when there is a large number of ROS compared to the anti-oxidants available. Some studies have suggested that analysis of gene expression of COCs might be used as an indirect predictor of oocyte quality and of the outcome of ART procedures, which could lead to distinct clinical applications (Hamamah et al., 2006; Assou et al., 2006, 2008; Hamel et

In the female reproductive system, ROS and anti-oxidants play physiological roles during folliculogenesis, oocyte maturation, lutheal regression and fertilization (Agarwal et al., 2006). For example, an increase in ROS production in granulosa cells (Jancar et al., 2007) and on oxidative damage to DNA marker (8-hydroxy-20-deoxyguanosine) levels in granulosa cells and COCs (Seino et al., 2002) was associated with lower fertilization, poor embryo quality and reduction of implantation rates. Nevertheless, oxidative stress also seems to be associated with the etiopathogenesis of reproduction, as is the case in endometriosis (Guerin et al., 2001; Van Langendonckt et al., 2002; Agarwal et al., 2003; Barcelos et al., 2008),

Considering this substantial involvement of ROS and oxidative stress in fertilization and reproduction modulation, it is accepted that anti-oxidant enzymes on COCs modulate oocyte maturation and might be related to specific conditions that limit the success of ART. Some studies have shown that superoxide dismutase (La Polt & Hong, 1995) might have inhibitory effects on aromatase, suggesting a potential correlation between gene expression

Abreu LG, Romão GS, Dos Reis RM, Ferriani RA, De Sá MF, De Moura MD. Reduced

Agarwal A, Said TM, Bedaiwy MA, et al. Oxidative stress in an assisted reproductive

Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology

Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction

Albertini DF, Barrett SL. Oocyte-somatic cell communication. Reproduction. 2003 61:49-54.

aromatase activity in granulosa cells of women with endometriosis undergoing assisted reproduction techniques. Gynecol Endocrinol. 2006 Aug;22(8):432-6. Agarwal A, Gupta S, Sharma R. Role of oxidative stress in female reproduction. Reprod Biol

idiopathic infertility and polycystic ovary syndrome (Gonzalez et al., 2006).

of one of the major anti-oxidant enzymatic system and aromatase expression.

techniques setting. Fertil Steril. 2006; 86(3):503-12.

of human reproduction. Fertil Steril. 2003, 79(4):829-843.

al., 2008; Tesfaye et al., 2009; Haouzi & Hamamah, 2009).

(COCs).

**8. References** 

Endocrinol. 2005; 3:28.

2001;122:497–506.

2003.

development of a follicle that holds a mature oocyte (Costa et al., 2004; Speroff & Fritz, 2005; Silva et al., 2008). Androgens, for instance, are necessary at low concentrations at the very beginning of follicle development, as a substrate for estradiol production. According to the two cells theory, theca cells convert C21 components (cholesterol) to androgen, which is a substrate for the aromatase of granulosa cells that converts androgens (C19) to estrogens (C18). The transformation of an androgenic environment to an estrogenic one is crucial in order to produce an oocyte capable of ovulation (Speroff & Fritz, 2005). In granulosa cells, aromatase plays an essential role in folliculogenesis and in estradiol production and its expression increases with follicle development (Tetsuka &Hillier, 1997; Guet et al., 1999) under the influence of FSH (Speroff & Fritz, 2005). Therefore, aromatase is a crucial enzyme in granulosa cells which is responsible for the formation of an estrogenic follicle microenvironment, essential for development and maturation (Speroff & Fritz, 2005). Nevertheless, it is important to state that aromatase is the final point of the entire ovarian steroidogenic cascade and the only enzyme capable of converting androgens to estrogens. Therefore, if its activity is impaired, that specific follicle will have difficulty in acquiring a normal pre-ovulatory state.

Intra-follicle hormonal relations are essential for the success of the entire ovulatory process both in natural cycles and in cycles stimulated for ART. Regarding maturation, Costa et al. (2004) analyzed cycles stimulated with exogenous gonadotropins without using a GnRH analogue and found that the follicles that held mature oocytes presented an increase in the progesterone/testosterone (P/T) ratio), in the progesterone/estradiol (P/E2) ratio and in the estradiol/testosterone (E/T) ratio in follicular fluid when compared to immature oocytes, suggesting a decrease in C21 to C19 conversion, but not in aromatase activity. Silva et al. (2008) analyzed these same ratios in follicles of women submitted to stimulated cycles using a GnRH analogue and observed that the action of the analogue remained intact and its most important effect was a decrease in intra-follicle androgen, with higher rates of fertilization and maturation.

In vitro studies using granulosa cell culture of women with endometriosis submitted to ovarian hyperstimulated cycles showed that these cells present impaired aromatase activity. Harlow et al. (1996) investigated aromatase activity in patients with minimal and mild endometriosis using granulosa cell culture in which estrogen production was evaluated after adding testosterone to the culture medium. They found a decrease in aromatase activity in patients with endometriosis compared to control. Researchers from the same group (Cahill et al., 2003) using the same technique found a lower sensitivity to LH in granulosa cells of patients with endometriosis.

Abreu et al. (2006) found a reduction of estradiol production in *in vitro* luteinized mural granulosa cells of women with endometriosis, after 24 hours of cell culture. Under baseline conditions or when the culture medium was supplemented with a lower concentration of testosterone (2x10-6M), estradiol production was lower in the endometriosis group. However, when the concentration of testosterone (an aromatase precursor) added to the culture medium was increased (2x10-5M), there was no difference between the endometriosis and control groups concerning estradiol production. In another study performed by Abreu et al. (2009) no difference in aromatase gene expression (CYP19A1) was observed in luteinized mural cells of women with endometriosis and controls submitted to

development of a follicle that holds a mature oocyte (Costa et al., 2004; Speroff & Fritz, 2005; Silva et al., 2008). Androgens, for instance, are necessary at low concentrations at the very beginning of follicle development, as a substrate for estradiol production. According to the two cells theory, theca cells convert C21 components (cholesterol) to androgen, which is a substrate for the aromatase of granulosa cells that converts androgens (C19) to estrogens (C18). The transformation of an androgenic environment to an estrogenic one is crucial in order to produce an oocyte capable of ovulation (Speroff & Fritz, 2005). In granulosa cells, aromatase plays an essential role in folliculogenesis and in estradiol production and its expression increases with follicle development (Tetsuka &Hillier, 1997; Guet et al., 1999) under the influence of FSH (Speroff & Fritz, 2005). Therefore, aromatase is a crucial enzyme in granulosa cells which is responsible for the formation of an estrogenic follicle microenvironment, essential for development and maturation (Speroff & Fritz, 2005). Nevertheless, it is important to state that aromatase is the final point of the entire ovarian steroidogenic cascade and the only enzyme capable of converting androgens to estrogens. Therefore, if its activity is impaired, that specific follicle will have difficulty in acquiring a

Intra-follicle hormonal relations are essential for the success of the entire ovulatory process both in natural cycles and in cycles stimulated for ART. Regarding maturation, Costa et al. (2004) analyzed cycles stimulated with exogenous gonadotropins without using a GnRH analogue and found that the follicles that held mature oocytes presented an increase in the progesterone/testosterone (P/T) ratio), in the progesterone/estradiol (P/E2) ratio and in the estradiol/testosterone (E/T) ratio in follicular fluid when compared to immature oocytes, suggesting a decrease in C21 to C19 conversion, but not in aromatase activity. Silva et al. (2008) analyzed these same ratios in follicles of women submitted to stimulated cycles using a GnRH analogue and observed that the action of the analogue remained intact and its most important effect was a decrease in intra-follicle androgen, with higher rates of fertilization

In vitro studies using granulosa cell culture of women with endometriosis submitted to ovarian hyperstimulated cycles showed that these cells present impaired aromatase activity. Harlow et al. (1996) investigated aromatase activity in patients with minimal and mild endometriosis using granulosa cell culture in which estrogen production was evaluated after adding testosterone to the culture medium. They found a decrease in aromatase activity in patients with endometriosis compared to control. Researchers from the same group (Cahill et al., 2003) using the same technique found a lower sensitivity to LH in

Abreu et al. (2006) found a reduction of estradiol production in *in vitro* luteinized mural granulosa cells of women with endometriosis, after 24 hours of cell culture. Under baseline conditions or when the culture medium was supplemented with a lower concentration of testosterone (2x10-6M), estradiol production was lower in the endometriosis group. However, when the concentration of testosterone (an aromatase precursor) added to the culture medium was increased (2x10-5M), there was no difference between the endometriosis and control groups concerning estradiol production. In another study performed by Abreu et al. (2009) no difference in aromatase gene expression (CYP19A1) was observed in luteinized mural cells of women with endometriosis and controls submitted to

normal pre-ovulatory state.

and maturation.

granulosa cells of patients with endometriosis.

ART. However, data obtained by the analysis of gene expression of mural granulosa cells of patients with endometriosis cannot be necessarily extrapolated to cumulus oophorus cells (COCs).

We found evidence that COCs might contribute to oocyte cytoplasmic maturation (Tanghe et al., 2002) through a net of gap junctions between COCs and between these and the oocyte (Furger et al., 1996). Nevertheless, the presence of COCs is important for fertilization to occur (Tanghe et al., 2002) because it attracts selected spermatozoa and promotes their capacitation and penetration. On the other hand, it should be emphasized that COCs protect the oocyte against apoptosis induced by oxidative stress (Tatemoto et al., 2000), which occurs when there is a large number of ROS compared to the anti-oxidants available. Some studies have suggested that analysis of gene expression of COCs might be used as an indirect predictor of oocyte quality and of the outcome of ART procedures, which could lead to distinct clinical applications (Hamamah et al., 2006; Assou et al., 2006, 2008; Hamel et al., 2008; Tesfaye et al., 2009; Haouzi & Hamamah, 2009).

In the female reproductive system, ROS and anti-oxidants play physiological roles during folliculogenesis, oocyte maturation, lutheal regression and fertilization (Agarwal et al., 2006). For example, an increase in ROS production in granulosa cells (Jancar et al., 2007) and on oxidative damage to DNA marker (8-hydroxy-20-deoxyguanosine) levels in granulosa cells and COCs (Seino et al., 2002) was associated with lower fertilization, poor embryo quality and reduction of implantation rates. Nevertheless, oxidative stress also seems to be associated with the etiopathogenesis of reproduction, as is the case in endometriosis (Guerin et al., 2001; Van Langendonckt et al., 2002; Agarwal et al., 2003; Barcelos et al., 2008), idiopathic infertility and polycystic ovary syndrome (Gonzalez et al., 2006).

Considering this substantial involvement of ROS and oxidative stress in fertilization and reproduction modulation, it is accepted that anti-oxidant enzymes on COCs modulate oocyte maturation and might be related to specific conditions that limit the success of ART. Some studies have shown that superoxide dismutase (La Polt & Hong, 1995) might have inhibitory effects on aromatase, suggesting a potential correlation between gene expression of one of the major anti-oxidant enzymatic system and aromatase expression.
