Infertility is a disease defined as "the inability to conceive following 12 or more months of unprotected sex before an investigation is undertaken unless the medical history and physical findings dictate earlier evaluation and and treatment [
67]. The prevalence of female infertility ranges from 7% to 28%, depending on the age of the woman. In general, an estimated 84% of couples conceive after 1 year of intercourse, and 92% of the couples conceive after 2 years [
68]. Although the frequency and origin of different forms of infertility varies, 40%–50% of the etiology of infertility studied is due to female causes [
69].
A primary diagnosis of male factor infertility is made in 30% of infertile couples. Combined female and male factor infertility is responsible for 20%–30% of cases. Finally, unexplained infertility affects 15% of couples [
70]. If the results of a standard infertility examination are normal, a diagnosis of unexplained or idiopathic infertility is assigned [
70]. Data from the National Survey for Family Growth indicate that the number of women with impaired fecundity has increased from 1982 to 1995, an increase of 35% in the number of women. Approximately 1.3 million American couples receive medical advice or treatment for infertility every year [
71]. OS has a role in etiopathogenesis of endometriosis, tubal factor infertility, and unexplained infertility. Impact of OS on ART is discussed in further sections.
2.2 Cytokines, oxidative stress and female reproduction
The control of ovarian stromal cells and germ cell function is a diverse paradigm and oxidative stress may be one of the modulators of ovarian germ cell and stromal cell physiology. A number of autocrine and paracrine factors affect the modulation of various ovarian functions and steroidogenesis. Cytokines are polypeptides or glycoproteins secreted into the extra cellular compartment by the leukocytes [
75]. Mammalian ovulation or follicular rupture was proposed to result from the vascular changes and the proteolytic cascade [
54]. The cross talk between these two cascades is mediated by cytokines, vascular endothelial growth factor (VEGF), and ROS (both reactive nitrogen and oxygen radicals). Interleukin-1β causes nitrite to accumulate in rat ovarian dispersates, demonstrating the close interaction between cytokines and NOS [
76]. OS and cytokines are proposed to be interlinked and act as intercellular and intracellular messengers in the ovary. A number of investigators have investigated the synthesis of NOS and ROS in the ovaries [
21,
55,
58].
Defective placentation leads to placental hypoxia and reperfusion injury due to ischemia and the resultant OS triggers the release of cytokines and prostaglandins, which results in endothelial cell dysfunction and plays an important role in the development of pre-eclampsia [
77,
78]. TNF-α a plasma cytokine, has been demonstrated to cause the endothelial cell injury [
79]. A link between OS and expression of cytokine receptors in the cytotrophoblast, vascular smooth muscle cells and endometrial cells has also been proposed, further establishing that hyperactivation of ROS may result in pre-eclampsia [
80].
The activation of mononuclear phagocytes can be triggered in endometriosis by a number of factors including damaged red blood cells and the apoptotic endometrial cells. A positive correlation between concentrations of tumor necrosis factor (TNF)-α in the peritoneal fluid and endometriosis has been reported [
75]. Cytokines released by the macrophages influence the redox status of the ectopic endometrium in patients with endometriosis [
81]. Superoxide dismutase, glutathione peroxidase activity and lipid peroxidation levels were measured in ectopic endometrial tissue obtained from ovarian endometriomas. Superoxide dismutase activity was found to be significantly higher in the ectopic endometrium than in eutopic endometrium, and a positive correlation was seen between malondialdehyde levels and plasma 17-beta estradiol levels. TNF-α has been shown to cause up regulation of expression of Manganese (Mn) superoxide dismutase in the endometrium in vitro [
82]. The antioxidant MnSOD neutralizes superoxide anions generated by cytokine TNF-α. This is a self protective mechanism against TNF-α induced oxidative stress. Estrogen and progesterone withdrawal leads to stimulation of prostaglandin F2α production via ROS-induced NFkappa β activation [
83]. The mechanism of menstruation is unclear, and activation of the transcription factor NFkappa β may be a piece in the puzzle.
Ovarian epithelial cancer is the most common type of ovarian cancer. Ovarian epithelial inflammation has been suggested as an etiological factor in ovarian epithelial cancer [
11,
84]. The mechanisms that bring about follicular rupture result in the exposure of the ovarian surface epithelial cells to deleterious agents (e.g. free radicals and TNF-α) [
85,
86]. Thus, incessant ovulation and its complex articulation by OS, inflammation and cytokines repeated cyclically may be involved in the etiopathogenesis of ovarian cancer [
86]. Factors that inhibit ovulation such as oral contraceptives reduce the risk of epithelial ovarian cancer [
87,
88]. Recent studies point towards a role of genes active in the process of metabolism of oxidation products, in the etiology of ovarian cancer [
89].
2.3 Reactive oxygen species and mediators of angiogenesis
Angiogenesis is a pathophysiological process involving formation of blood vessels from preexisting vessels. The induction of angiogenesis occurs when there is a deficiency of oxygen in tissues. This process of neovascularization results from hypoxia and induction of various angiogenic factors, and it has a role to play in physiological processes such as follicular development, endometrial growth, embryo development, growth of placental vessels and wound repair [
90,
91]. Angiogenesis is important for cyclical regeneration of endometrium in the menstrual cycle. A complex cytokine influence at the maternal-fetal interface creates the conditions that are necessary to support embryo implantation in the endometrium [
92,
93]. Any imbalance between the cytokines and angiogenesis factors could result in implantation failure and pregnancy loss [
94]. Critical changes occur in the vascular system, and these changes accompany follicular growth. Follicular growth, selection of dominant follicle, corpus luteum formation, endometrial differentiation and embryo formation are key processes dependent on neovascularization [
90,
95]. As the endometrium grows in the menstrual cycle, vessel regeneration occurs (i.e. spiral arterioles and capillaries) [
96]. Estrogens promote angiogenesis in the endometrium by controlling the expression of factors such as VEGF [
97]. ROS generated from NADP (H) oxidase is critical for VEGF signaling in vitro and angiogenesis in vivo [
98]. Small amounts of ROS are produced from endothelial NADP (H) oxidase activated by growth factors and cytokines.
ROS that are generated in and around the vascular endothelium may play a role in normal cellular signaling mechanisms. They may also be an important causative factors in endothelial dysfunction that leads to the development of atherosclerosis, diabetes complications and ischemia perfusion injury [
98,
99]. The molecular mechanism by which the oxidant status of cells modulates angiogenesis is not completely understood. As our understanding the role ROS-induced angiogenesis plays in atherosclerosis and myocardial angiogenesis grows, future studies should investigate the role ROS plays in the angiogenesis in the female reproductive tract.
2.5 ROS and endometriosis
Increased generation of ROS by activated peritoneal macrophages has been reported in the peritoneal fluid [
102]. Conflicting results were reported in further studies with large patient numbers, which failed to demonstrate an antioxidant or oxidant balance [
74,
103]. ROS levels in peritoneal fluid of patients with endometriosis were not significantly higher than controls.
An increased titer of autoantibodies related to OS has been reported in women with endometriosis resulting in an increase in serum autoantibody titers to oxidatively modified low density lipoproteins [
104]. An OS-induced increase in autoantibody titers in the peritoneal fluid has been demonstrated in women with endometriosis. Elevated levels of the marker of lipid peroxidation lysophophatidyl choline, a potent chemotactic factor for monocytes/T-lymphocytes, were seen in the peritoneal fluid of women with endometriosis [
105]. Non-terminal oxidation may have a role in the pathophysiology of endometriosis. Minimally oxidized low density lipoprotein (LDL) (M-LDL) is present in peritoneal fluid of women with endometriosis in place of the terminally oxidized LDL (Ox-LDL) [
106]. The ratio of lysophosphatidyl choline, a breakdown product of Ox-LDL, to phosphatidyl choline suggests M-LDL rather than Ox-LDL. Modest levels of OS induced proliferation of endometrial stromal cells in vitro, was inhibited by antioxidants [
107]. RU486, a potent antiprogestational agent with antioxidant activity also decreased proliferation of epithelial and stromal cells [
108].
2.6 Reactive oxygen species and the ovary
Markers of oxidative stress such as superoxide dismutase, Cu-Zn superoxide dismutase, Mn superoxide dismutase, glutathione peroxidase, γ glutamyl synthetase and lipid peroxides have been investigated by immunohistochemical localization, m-RNA expression and thiobarbituric acid method [
4,
14,
41]. The expression of various biomarkers of OS has been demonstrated in normal cycling human ovaries [
13,
14]. All follicular stages have been examined for SOD expression including primordial, primary, preantral and nondominant antral follicles in follicular phase, dominant follicles and atretic follicles [
14]. ROS may have a regulatory role in oocyte maturation, folliculogenesis, ovarian steroidogenesis and luteolysis. There is a delicate balance between ROS and antioxidant enzymes in the ovarian tissues. The antioxidant enzymes neutralize ROS production and protect the oocyte and embryo.
The presence of superoxide dismutase in the ovary, revealed intense staining by immunohistochemistry in the theca interna cells in the antral follicles [
13]. Antibody to Ad4-binding protein (Ad4BP) was utilized to localize Ad4BP in the nuclei of theca and granulosa cells. Ad4BP is a steroidogenic transcription factor that induces transcription of the steroidogenic P450 enzyme. Thus, it controls steroidogenesis in the ovaries. The correlation between Ad4BP and superoxide dismutase expression suggests an association between OS and ovarian steroidogeneis [
14].
Both human granulosa and luteal cells respond to hydrogen peroxide with an extirpation of gonadotropin action and inhibition of progesterone secretion [
11]. The production of both progesterone and estradiol hormones is reduced when hydrogen peroxide is added to a culture of human chorionic gonadotropin-stimulated luteal cells. Hydrogen peroxide lowers both cAMP dependent and non-cAMP dependent steroidogenesis [
109]. The role of hCG (human chorionic gonadotropin) in the expression of the antioxidant enzyme superoxide dismutase (SOD) has been investigated. Corpora lutea collected from patients at hysterectomy and surgery for ectopic pregnancy were studied [
14]. The Cu-Zn SOD expression in the corpora lutea paralleled levels of progesterone and these levels rose from early to mid luteal phase and decreased during the regression of the corpus luteum. However, in the corpus luteum from pregnant patients, the mRNA expression for Cu-Zn superoxide dismutase was significantly higher than that in midcycle corpora lutea. This enhanced expression of luteal Cu-Zn SOD may be due to hCG and hCG may have an important role in maintenance of corpus luteal function in pregnancy.
Levels of three oxidative stress biomarkers, conjugated dienes, lipid hydroperoxide and thiobarabituric acid were determined in preovulatory follicles. Concentration gradient was found to exist as levels of all three markers were significantly lower in the follicular fluid compared with serum levels [
15]. The preovulatory follicle has a potent antioxidant defense, which is depleted by the intense peroxidation [
15]. Glutathione peroxidase may also maintain low levels of hydroperoxides inside the follicle and thus play an important role in gametogenesis and fertilization [
42].
2.7 Nitric Oxide synthase in female reproduction
The production of a viable oocyte is modulated by a complex interaction of endocrine, paracrine and autocrine factors leading to follicular maturation, granulosa cell maturation, ovulation and luteinization. Many hormonal and paracrine factors determine oocyte competence and embryo quality. Steroid hormones and local autocrine and paracrine factors influence ovarian stromal cells. The gonadotropins act through complex, multiple local signaling pathways. Cyclic AMP is thought to be the second messenger to bring about the effect of luteinizing hormone and follicular stimulating hormone [
110]. In turn, cyclic AMP may activate other signaling pathways. Cyclic guanosine monophosphate (cGMP)-a cyclic nucleotide has also been proposed as a second messenger pathway. The effects of NO are proposed to be mediated through cGMP as a second messenger or by generation of ROS resulting from interaction of NO with superoxide radicals [
111].
NO generated by macrophages in response to invading microbes acts as an antimicrobial agent [
21]. Neurons, blood vessels and cells of the immune system are integral parts of the reproductive organs, and in view of the important functional role that NO plays in those systems, it is highly likely that NO is an important regulator of the biology and physiology of the reproductive system. NO has established itself as a polyvalent molecule that plays a decisive role in regulating multiple functions within the female as well as male reproductive system [
21]. As a final immune effector, NO generated by inducible NO synthase, kills pathogens and abnormal cells but may play a detrimental role by damaging normal host tissue and cells, especially when inducible NO synthase is persistently expressed [
20].
2.9 Nitric oxide synthase, endometrium, and myometrium
Expression of endothelial and inducible NO synthase have been demonstrated in the human endometrium [
115], and the endometrial vessels [
116]. Endothelial NO synthase, originally identified in vascular endothelial cells, is distributed in glandular surface epithelial cells in the human endometrium. NO also regulates the microvasculature of the endometrium and is important in menstruation.
Endothelial NOS like immunoreactivity has been reported in the endothelial cells lining the vessels, endometrium and endometrial glandular epithelial cells and myometrium [
117]. Inducible NOS like immunoreactivity was detected in decidualised stromal cells and also expressed in tissues from first trimester of pregnancy. Thus, NO has a role to play in decidualisation of the endometrium and preparation of the endometrium for implantation.
Highest levels of expression of endothelial NOS mRNA have been reported in the late secretory phase of the endometrium [
115]. In addition, NO might participate in the regulation of uterine contraction [
118]. In normal fertile woman, the contractions increase throughout the proliferative and periovulatory phases, and decrease in the secretory phase. From this point, NO is synergistic with progesterone and might relax uterine contraction in the secretory phase in a paracrine fashion. Nitric oxide regulates the endometrial, myometrial and microvascular functions in the uterus by paracrine functions. The effects of the antiprogestational agent, Mifepristone on the endothelial NOS expression in the endometrium were investigated during the implantation phase [
119]. Mifepristone inhibited the endometrial glandular epithelial eNOS expression and did not affect the endothelilal eNOS. The results supported the role of epithelial eNOS in endometrial receptivity in the perimplantation period. Significant increase in the levels of serum metabolites of NOS amongst users of progestin only contraceptives has been reported [
120]. A positive correlation was also demonstrated between NO levels and prolonged/heavy bleeding. Thus NO may be involved in the pathophysiology of menorrhagia.
NO plays a significant role in pregnancy and labor. Expression of inducible NOS was highest in patients with preterm pregnancy and not in patients in term labor. The expression of these enzymes decreased by 75% at term and was barely detectable in preterm in labor patients or term labor patients [
121] reiterating that NO has a role in maintenance of uterine quiescence. However other authors have reported conflicting results. Induction of iNOS expression was demonstrated in fetal membranes in labour and in in-vitro studies [
122]. Higher NO metabolite concentrations were demonstrated in amniotic fluid collected from women in labor than in non-laboring patients, both at term (15.4 ± 1.6 vs. 6.8 ± 0.6 microM/mg creatinine) and preterm (16.7 ± 2.0 vs. 7.0 ± 0.8 microM/mg creatinine) [
123].
2.10 Nitric oxide synthase and endometriosis
Endometriosis is found in about 35% of infertile women who have laparoscopy as part of their infertility workup [
71]. Production of ROS by peritoneal fluid mononuclear cells was reported to be associated with endometriosis [
75]. Low levels of NO are important in ovarian function and implantation and cause relaxation of oviduct musculature [
112]. High levels of NO are reported as having deleterious effects on sperm motility, toxic to embryos and inhibit implantation [
124,
125]. In vitro fertilization, a process that avoids contact of gametes and embryos with potentially toxic peritoneal and oviductal factors associated with endometriosis (e.g., NOS, ROS) improves the chances of conception in these women. NO is a free radical with deleterious effects and is an important bioregulator of apoptosis [
126]. Activation of polymorphonuclear leucocytes and macrophages leads to increased production of ROS [
102]. Increase in number and activity of macrophages is accompanied by release of more cytokines and other immune mediators, such as NO. This was initially implicated in low-grade inflammation, while elevated peritoneal NO is consistent with the increased number and activity of macrophages [
20]. High levels of NO, such as those produced by macrophages, can negatively influence fertility in several ways. Changes in peritoneal fluid, an environment that hosts the process of ovulation, gamete transportation, sperm oocyte interaction, fertilization, and early embryonic development, might affect all these steps of reproduction [
2,
20,
127]. Studies investigating the association of nitric oxide levels and lipid peroxides and reactive oxygen species in peritoneal fluid did not find any significant difference in patients with or without endometriosis [
103,
128,
129] Conflicting results were obtained in studies conducted by Szczepanska et al [
2]. The total antioxidant capacity reduced and the individual antioxidant enzymes like superoxide dismutase were significantly lower in the peritoneal fluid of women with endometriosis-associated infertility. The lipid peroxide levels were the highest amongst patients with endometriosis suggesting role of ROS in the development of the disease process [
2]. There is a cyclical expression of mRNA of NOS in the epithelial glands of the human endometrium. Higher amounts of NO and NOS are seen in the endometrium of women with endometriosis [
28,
130,
131]. NOS expression in the ectopic endometrium of patients with adenomyosis is continuous throughout the menstrual cycle [
132].
Peritoneal fluid NO levels, peritoneal macrophage NOS activity, and peritoneal macrophage inducible NOS protein expression has been examined in women with endometriosis-associated infertility. Peritoneal macrophages express higher levels of NOS, have higher NOS enzyme activity, and produce more NO in response to immune stimulation in vitro [
23]. High levels of NO adversely affect sperm, embryos, implantation, and oviductal function, indicating that reduction in the peritoneal fluid NO production or blocking NO effects may improve fertility in women with endometriosis [
23]. However, generation of peroxynitrite by ectopic endometrium has been reported in patients with adenomyosis. Expressions of endothelial and inducible NO synthase and peroxynitrite generation was markedly reduced after GnRH agonist therapy, supporting their potential role in the pathophysiology of adenomyosis [
132]. Serum NO levels are suppressed by GnRH agonist and upregulated by gonadotropin stimulation during controlled ovarian stimulation in female partners from couples with male factor infertility [
133]. Maximal levels were measured at the time of ovulation in the same study. Elevated NO production was not demonstrated in patients with ovarian hyperstimulation.
Increased levels of NO were demonstrated in the peritoneal fluid of patients with endometriosis [
20,
23]. It has also been hypothesized that ROS may have a role in formation of adhesions associated with endometriosis [
134]. Patients with endometriosis show a higher 8-hydroxy 1-deoxyguanosine index compared to patients with other causes of infertility, such as tubal, male factor or idiopathic causes [
52].
Expression of NOS is elevated in patients with endometriosis, and a common polymorphism of exon 7 at nucleotide 894 in the endothelial NOS gene may be associated with endometriosis [
135]. Hence variations in the expression of the eNOS gene may be involved in endometrial angiogenesis and thus modulate the process of endometriosis.
Expression of endothelial NO synthase in the endometrium of patients with endometriosis or adenomyosis is persistently marked throughout the menstrual cycle [
132]. Many investigators have reported increased expression of endothelial NOS in the glandular endometrium in patients with endometriosis [
28,
130]. Inducible NOS isoform is elevated in tissues of patients with endometriosis [
131]. The endometrial development affects embryo implantation, and inconsistent development between endometrium and embryo could impede embryo implantation. Nitric oxide affects fecundity in endometriosis and adenomyosis [
136]. Significant differences are seen in the uterine hyperperistalsis and dysperistalsis in patients with endometriosis compared with the control groups, and this may be responsible for disturbed sperm transport and reduced fertility [
137].
Various cytokines secreted from endometrial cells, immune cells, or macrophages stimulate endothelial NO synthase to release NO [
3,
28,
136]. These abnormal immune responses might eventually stimulate macrophages and/or endometrial cells to persistently produce a large amount of NO and inhibit implantation [
138]. Increased expression of endothelial NO synthase has been reported throughout the menstrual cycle in the endometrium of women with endometriosis [
139].
2.11 Nitric oxide synthase and the ovary
Ovarian follicullogenesis not only involves gonadotropins and the steroids, but it also involves local autocrine and paracrine factors. Nitric oxide radical is one of the local factors involved in ovarian follicullogeneis and steroidogenesis. Nitric oxide acts through activation of various iron containing enzymes. It binds to the heme containing enzyme guanylate cyclase, which activates the cyclic nucleotide cyclic-GMP [
110]. Plasma concentrations of nitrate monitored during the follicular cycle, have revealed peak levels at ovulation [
133,
140]. Nitric oxide inhibits ovarian steroidogenesis [
52]. The presence of endothelial NO synthase in human corpora lutea and the expression has been reported in the mid and early luteal phase and to a lesser extent in the late luteal [
53]. Nitric oxide inhibits steroidogenesis in the corpus luteum and has luteolytic action mediated through increased prostaglandins and by apoptosis [
53,
141].
Follicular fluid NO seems to be produced by either endothelial NO synthase or induced NO synthase. However, in the normal physiological conditions follicular fluid NO seems to be synthesized from granulosa cells by endothelial NO synthase, since in isolated human follicular cells at least 90% of cells are granulosa cells even though macrophages and lymphocytes are present as well. In patients undergoing IVF, a positive correlation was determined between follicular fluid nitrate/nitrite levels and the follicular volume as well as the serum estradiol concentration [
142]. In contrast to these findings, Manau et al, found no association between follicular fluid nitrite/nitrate levels and parameters of ovarian response [
143]. Biomarkers like serum nitric oxide measurements cannot be used as predicting success with IVF [
143,
144]. Serum nitrate concentration may not be a good biomarker because of the short half-life of NO. Follicular blood flow was found to be a better prognostic factor for predicting successful outcomes with IVF than follicular NO levels [
138]. Follicular fluid NO levels were altered in patients with infertility associated diseases. NO follicular fluid levels were significantly higher in patients with endometriosis or hydrosalpinx compared to patients with tubal obstruction [
29]. No correlation was reported between the follicular NO levels and follicle maturity or follicle quality.
Some studies have demonstrated the relationship between NO concentrations in follicular growth and programmed follicular cell death (apoptosis). Folliculogenesis involves the participation of both growth of the follicle and apoptosis. Nitric oxide regulates both of these processes [
21]. Sugino et al studied the role of nitric oxide in follicular atresia and apoptosis, in patients undergoing IVF and found that the smaller follicles had significantly elevated percentage of apoptotic granulosa cells with nuclear fragmentation [
58]. Low concentrations of NO may prevent apoptosis, however pathologically high concentrations of NO, as well as increased superoxide generation by NO synthase due to lack of arginine, may promote cell death by peroxynitrite generation [
21]. Nitric oxide involvement in various ovarian functions has been suggested. The presence of NO in the follicular fluid and the expression of NO synthase in follicles and corpus luteum have been reported [
19,
141,
143,
145].
Plasma concentration of NO was shown to increase in the follicular phase compared with the secretory phase and peaked at midcycle [
140]. Nitric oxide elicited a positive effect on women with poor ovarian response compared to controlled ovarian stimulation [
146]. Upregulated NO is harmful to implantation and pregnancy among patients with tubal factor infertility after controlled ovarian stimulation [
147]. Serum NO levels were elevated amongst nonpregnant patients with tubal or peritoneal factor infertility [
124].
Follicular fluid NO level is not associated with maturity or quality of oocyte and no significant differences were seen in concentrations of NO of follicular fluid among large, medium, or small follicle size. Higher TNF-α concentrations in follicular fluid correlated with poor oocyte quality [
29]. Whereas, follicular fluid nitrite or nitrate levels were significantly lower in follicles containing mature oocytes that fertilized compared with those that did not [
148]. Follicular NO has been reported to correlate negatively with embryo quality and the rate of embryo cleavage [
124,
147,
148]. The beneficial effects of NO donors in patients with intrauterine growth retardation (IUGR) and inhibition of pre-term labor has been studied [
149,
150]. Using a nitroglycerine (NTG) patch, which is a NO donor, did not significantly affect the final outcome in patients undergoing in-vitro fertilization. In addition, neither placebo nor the nitroglycerine patch improved the flow resistance in the uterine artery [
22]. NO donors and elevated serum NO was associated with implantation failure resulting in decreased fertility [
138].