9.1. Endometriosis
Endometriosis is a benign, estrogen-dependent, chronic gynecological disorder characterized by the presence of endometrial tissue outside the uterus. Lesions are usually located on dependent surfaces in the pelvis and most often affect the ovaries and cul-de-sac. They can also be found in other areas such as the abdominal viscera, the lungs, and the urinary tract. Endometriosis affects 6% to 10% of women of reproductive age and is known to be associated with pelvic pain and infertility [
73], although it is a complex and multifactorial disease that cannot be explained by a single theory, but by a combination of theories. These may include retrograde menstruation, impaired immunologic response, genetic predisposition, and inflammatory components [
74]. The mechanism that most likely explains pelvic endometriosis is the theory of retrograde menstruation and implantation. This theory poses that the backflow of endometrial tissue through the fallopian tubes during menstruation explains its extra-tubal locations and adherence to the pelvic viscera [
75].
Studies have reported mixed results regarding detection of OS markers in patients with endometriosis. While some studies failed to observe increased OS in the peritoneal fluid or circulation of patients with endometriosis [
76‐
78], others have reported increased levels of OS markers in those with the disease [
79‐
83]. The peritoneal fluid of patients have been found to contain high concentrations of malondialdehyde (MDA), pro-inflammatory cytokines (IL-6, TNF-alpha, and IL-beta), angiogenic factors (IL-8 and VEGF), monocyte chemoattractant protein-1 [
82], and oxidized LDL (ox-LDL) [
84]. Pro-inflammatory and chemotactic cytokines play a central role in the recruitment and activation of phagocytic cells, which are the main producers of both ROS and RNS [
82].
Non-enzymatic peroxidation of arachidonic acid leads to the production of F2-isoprostanes [
85]. Lipid peroxidation, and thus, OS in vivo [
83], has been demonstrated by increased levels of the biomarker 8-iso-prostaglandin F2-alpha (8-iso-PGF2-alpha) [
86‐
88]. Along with its vasoconstrictive properties, 8-iso-PGF2-alpha promotes necrosis of endothelial cells and their adhesion to monocytes and polymorphonuclear cells [
89]. A study by Sharma et al (2010) measured peritoneal fluid and plasma levels of 8-iso-PGF2-alpha in vivo of patients with endometriosis. They found that 8-iso-PGF2-alpha levels in both the urine and peritoneal fluid of patients with endometriosis were significantly elevated when compared with those of controls [
83]. Levels of 8-iso-PGF2-alpha are likely to be useful in predicting oxidative status in diseases such as endometriosis, and might be instrumental in determining the cause of concurrent infertility.
A collective term often used in reference to individual members of the HSP70 family is ‘HSP70’ [
90]. The main inducible forms of HSP70 are HSPA1A and HSPA1B [
91], also known as HSP70A and HSP70 B respectively [
90]. Both forms have been reported as individual markers of different pathological processes [
92].
Heat shock protein 70 B is an inducible member of HSP family that is present in low levels under normal conditions [
93] and in high levels [
94] under situations of stress. It functions as a chaperone for proteostatic processes such as folding and translocation, while maintaining quality control [
95]. It has also been noted to promote cell proliferation through the suppression of apoptosis, especially when expressed in high levels, as noted in many tumor cells [
94,
96‐
98]. As such, HSP70 is overexpressed when there is an increased number of misfolded proteins, and thus, an overabundance of ROS [
94]. The release of HSP70 during OS stimulates the expression of inflammatory cytokines [
93,
99] TNF-alpha, IL-1 beta, and IL-6, in macrophages through toll-like receptors (e.g. TLR 4), possibly accounting for pelvic inflammation and growth of endometriotic tissue [
99].
Another inducible form of HSP70 known as HSP70b′ has recently become of great interest as it presents only during conditions of cellular stress [
100]. Lambrinoudaki et al (2009) have reported high concentrations of HSP70b′ in the circulation of patients with endometriosis [
101]. Elevated circulating levels of HSP70b′ may indicate the presence of OS outside the pelvic cavity when ectopic endometrial tissue is found in distal locations [
101].
Fragmentation of HSP70 has been suggested to result in unregulated expression of transcription factor NF-kappa B [
102], which may further promote inflammation within the pelvic cavity of patients with endometriosis. Oxidants have been proposed to encourage growth of ectopic endometrial tissue through the induction of cytokines and growth factors [
103]. Signaling mediated by NF-kappa B stimulates inflammation, invasion, angiogenesis, and cell proliferation; it also prevents apoptosis of endometriotic cells. Activation of NF-kappa B by OS has been detected in endometriotic lesions and peritoneal macrophages of patients with endometriosis [
104]. N-acetylcysteine (NAC) and vitamin E are antioxidants that limit the proliferation of endometriotic cells [
105], likely by inhibiting activation of NF-kappa B [
106]. Future studies may implicate a therapeutic effect of NAC and vitamin E supplementation on endometriotic growth.
Similar to tumor cells, endometriotic cells [
107] have demonstrated increased ROS and subsequent cellular proliferation, which have been suggested to occur through activation of MAPK extracellular regulated kinase (ERK1/2) [
108]. The survival of human endometriotic cells through the activation of MAPK ERK 1/2, NF-kappa B, and other pathways have also been attributed to PG E2, which acts through receptors EP2 and EP4 [
109] to inhibit apoptosis [
110]. This may explain the increased expressions of these proteins in ectopic versus eutopic endometrial tissue [
109].
Iron mediates production of ROS via the Fenton reaction and induces OS [
111]. In the peritoneum of patients with endometriosis, accumulation of iron and heme around endometriotic lesions [
112] from retrograde menstruation [
113] up-regulates iNOS activity and generation of NO by peritoneal macrophages [
114]. Extensive degradation of DNA by iron and heme accounts for their considerable free radical activity. Chronic oxidative insults from iron buildup within endometriotic lesions may be a key factor in the development of the disease [
115].
Naturally, endometriotic cysts contain high levels of free iron as a result of recurrent cyclical hemorrhage into them compared to other types of ovarian cysts. However, high concentrations of lipid peroxides, 8-OHdG, and antioxidant markers in endometrial cysts indicate lipid peroxidation, DNA damage, and up-regulated antioxidant defenses respectively. These findings strongly suggest altered redox status within endometrial cysts [
111].
Potential therapies have been suggested to prevent iron-stimulated generation of ROS and DNA damage. Based on results from their studies of human endometrium, Kobayashi et al (2009) have proposed a role for iron chelators such as dexrazoxane, deferoxamine, and deferasirox to prevent the accumulation of iron in and around endometriotic lesions [
115]. Future studies investigating the use of iron chelators may prove beneficial in the prevention of lesion formation and the reduction of lesion size.
Many genes encoding antioxidant enzymes and proteins are recruited to combat excessive ROS and to prevent cell damage. Amongst these are Trx and Trx reductase, which sense altered redox status and help maintain cell survival against ROS [
116]. Total thiol levels, used to predict total antioxidant capacity (TAC), have been found to be decreased in women with pelvic endometriosis and may contribute to their status of OS [
81,
101]. Conversely, results from a more recent study failed to correlate antioxidant nutrients with total thiol levels [
117].
Patients with endometriosis tend to have lower pregnancy rates than women without the disease. Low oocyte and embryo quality in addition to spermatotoxic peritoneal fluid may be mediated by ROS and contribute to the subfertility experienced by patients with endometriosis [
118]. The peritoneal fluid of women with endometriosis contains low concentrations of the antioxidants ascorbic acid [
82] and GPx [
81]. The reduction in GPx levels was proposed to be secondary to decreased progesterone response of endometrial cells [
119]. The link between gene expression for progesterone resistance and OS may facilitate a better understanding of the pathogenesis of endometriosis.
It has been suggested that diets lacking adequate amounts of antioxidants may predispose some women to endometriosis [
120]. Studies have shown decreased levels of OS markers in people who consume antioxidant rich diets or take antioxidant supplements [
121‐
124]. In certain populations, women with endometriosis have been observed to have a lower intake of vitamins A, C [
125], E [
125‐
127], Cu, and Zn [
125] than fertile women without the disease [
125‐
127]. Daily supplementation with vitamins C and E for 4 months was found to decrease levels of OS markers in these patients, and was attributed to the increased intake of these vitamins and their possible synergistic effects. Pregnancy rates, however, did not improve [
126].
Intraperitoneal administration of melatonin, a potent scavenger of free radicals, has been shown to cause regression of endometriotic lesions [
128‐
130] by reducing OS [
129,
130]. These findings, however, were observed in rodent models of endometriosis, which may not closely resemble the disease in humans.
It is evident that endometriotic cells contain high levels of ROS; however, their precise origins remain unclear. Impaired detoxification processes lead to excess ROS and OS, and may be involved in increased cellular proliferation and inhibition of apoptosis in endometriotic cells. Further studies investigating dietary and supplemental antioxidant intake within different populations are warranted to determine if antioxidant status and/or intake play a role in the development, progression, or regression of endometriosis.
9.2. Polycystic ovary syndrome
Polycystic ovary syndrome is the most common endocrine abnormality of reproductive-aged women and has a prevalence of approximately 18%. It is a disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovaries [
131]. Clinical manifestations of PCOS commonly include menstrual disorders, which range from amenorrhea to menorrhagia. Skin disorders are also very prevalent amongst these women. Additionally, 90% of women with PCOS are unable to conceive.
Insulin resistance may be central to the etiology of PCOS. Signs of insulin resistance such as hypertension, obesity, and central fat distribution are associated with other serious conditions, such as metabolic syndrome, nonalcoholic fatty liver [
132], and sleep apnea. All of these conditions are risk factors for long-term metabolic sequelae, such as cardiovascular disease and diabetes [
133]. Most importantly, waist circumference, independent of body mass index (BMI), is responsible for an increase in oxLDL [
71]. Insulin resistance and/or compensatory hyperinsulinemia increase the availability of both circulating androgen and androgen production by the adrenal gland and ovary mainly by decreasing sex hormone binding globulin (SHBG) [
134].
Polycystic ovary syndrome is also associated with decreased antioxidant concentrations, and is thus considered an oxidative state [
135]. The decrease in mitochondrial O
2 consumption and GSH levels along with increased ROS production explains the mitochondrial dysfunction in PCOS patients [
136]. The mononuclear cells of women with PCOS are increased in this inflammatory state [
137], which occurs more so from a heightened response to hyperglycemia and C-reactive protein (CRP). Physiological hyperglycemia generates increased levels of ROS from mononuclear cells, which then activate the release of TNF-alpha and increase inflammatory transcription factor NF-kappa B. As a result, concentrations of TNF-alpha, a known mediator of insulin resistance, are further increased. The resultant OS creates an inflammatory environment that further increases insulin resistance and contributes to hyperandrogenism [
138].
Lifestyle modification is the cornerstone treatment for women with PCOS. This includes exercise and a balanced diet, with a focus on caloric restriction [
139]. However, if lifestyle modifications do not suffice, a variety of options for medical therapy exist. Combined oral contraceptives are considered the primary treatment for menstrual disorders. Currently, there is no clear primary treatment for hirsutism, although it is known that combination therapies seem to produce better results [
138].
9.3. Unexplained infertility
Unexplained infertility is defined as the inability to conceive after 12 months of unprotected intercourse in couples where known causes of infertility have been ruled out. It is thus considered a diagnosis of exclusion. Unexplained infertility affects 15% of couples in the United States. Its pathophysiology remains unclear, although the literature suggests a possible contribution by increased levels of ROS, especially shown by increased levels of the lipid peroxidation marker, MDA [
140,
141] in comparison to antioxidant concentration in the peritoneal cavity [
142]. The increased amounts of ROS in these patients are suggestive of a reduction in antioxidant defenses, including GSH and vitamin E [
76]. The low antioxidant status of the peritoneal fluid may be a determinant factor in the pathogenesis of idiopathic infertility.
N-acetyl cysteine is a powerful antioxidant with anti-apoptotic effects. It is known to preserve vascular integrity and to lower levels of homocysteine, an inducer of OS and apoptosis. Badaiwy et al (2006) conducted a randomized, controlled, study in which NAC was compared with clomiphene citrate as a cofactor for ovulation induction in women with unexplained infertility [
143]. The study, however, concluded that NAC was ineffective in inducing ovulation in patients in these patients [
143].
Folate is a B9 vitamin that is considered indispensable for reproduction. It plays a role in amino acid metabolism and the methylation of proteins, lipids, and nucleic acids. Acquired or hereditary folate deficiency contributes to homocysteine accumulation. Recently, Altmae et al (2010) established that the most important variation in folate metabolism in terms of impact is methyl-tetra-hydrofolate reductase (MTHFR) gene polymorphism 677C/T [
144]. The MTHFR enzyme participates in the conversion of homocysteine to methionine, a precursor for the methylation of DNA, lipids, and proteins. Polymorphisms in folate-metabolizing pathways of genes may account for the unexplained infertility seen in these women, as it disrupts homocysteine levels and subsequently alters homeostatic status. Impaired folate metabolism disturbs endometrial maturation and results in poor oocyte quality [
144].
More studies are clearly needed to explore the efficacy of antioxidant supplementation as a possible management approach for these patients.