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After completion of this article, the reader should be able to state that the causes of spontaneous and recurrent abortion are multifaceted, however, some of the causes may be preventable and also explain that the role of oxidative stress during pregnancy and adverse pregnancy outcomes has a basis in pathophysiology, although the role of oxidative stress and the treatment of oxidative stress during or before pregnancy remains speculative.

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Hypochlorous acid (HOCl) is generated by myeloperoxidase, using chloride and hydrogen peroxide as substrates. Here we demonstrate that HOCl alters metaphase-II mouse oocyte microtubules and chromosomal (CH) alignment which can be prevented by melatonin. Metaphase-II mouse oocytes, obtained commercially, were grouped as: control, melatonin (150, 200nmol/mL), HOCl (10, 20, 50, and 100nmol/mL), and HOCl (50nmol/mL) pretreated with 150 and 200 nmol/mL of melatonin. Microtubule and CH alignment was studied utilizing an indirect immunofluorescence technique and scored by two observers. Pearson chi-square test and Fisher's exact test were used to compare outcomes between controls and treated groups and also among each group. Poor scores for the spindle and chromosomes increased significantly at 50nmol/mL of HOCl (P<0.001). Oocytes treated with melatonin only at 150 and 200 nmol/mL showed no changes; significant differences (P<0.001) were observed when oocytes exposed to 50nmol/mL of HOCl were compared to oocytes pretreated with 200 nmol/mL melatonin. Fifty percent of the oocytes demonstrated good scores, both in microtubule and CH alterations, when pretreated with melatonin at 150 nmol/mL compared to 0% in the HOCl-only group. HOCl alters the metaphase-II mouse oocyte spindle and CH alignment in a dose-dependant manner, which might be a potential cause of poor oocyte quality (e.g., in patients with endometriosis). Melatonin prevented the HOCl-mediated spindle and CH damage, and therefore, may be an attractive therapeutic option to prevent oocyte damage in endometriosis or inflammatory diseases where HOCl levels are known to be elevated.

Here we show that hydroxyl radical (•OH) generated through the Fenton reaction alters metaphase-II mouse oocyte microtubules (MT) and chromosomal alignment (CH). Metaphase–II mouse oocytes, obtained commercially, were grouped as follows: control, hydrogen peroxide (H2O2), Fe (II), and combined (Fe (II) + H2O2) treatments. After 7–10 minutes of incubation, at 37 °C, MT and CH were evaluated on fixed and stained oocytes and scored by two blinded observers. Pearson Chi-square test, Fisher's Exact test were used to compare outcomes between controls and treated groups, and also amongst each group. Our results showed that poor scores for MT and CH increased significantly in oocytes treated with combination of H2O2 and Fe(II) (p <0.001); oocytes treated with H2O2 alone or Fe(II) alone showed no or little changes compared to control. Comparison of oocyte groups that received increasing concentrations of H2O2 and fixed amount of Fe (II) showed that 70 – 80 % demonstrated poor scores both in MT and CH when pretreated with 5 μM H2O2, and increased up to 90–100% when treated with 10–20 μM of H2O2. Hydroxyl radical generated by H2O2 driven Fenton reaction deteriorates the metaphase-II mouse oocyte spindle and CH alignment, which is thought to be a potential cause of poor oocyte quality. Thus, free iron and/or ROS scavengers could attenuate the •OH-mediated spindle and chromosomal damage, thereby serving as a possible approach for further examination as a therapeutic option in inflammatory states.

Overproduction of hypochlorous acid (HOCl) has been associated with the development of variety of disorders such as inflammation, heart disease, pulmonary fibrosis and cancer through its ability to modify different biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl with heme destruction and free iron release from hemoglobin and RBCs. Our current finding extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl) a common pharmacological form of vitamin B12. Cyanocobalamin is a water soluble vitamin which plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that while Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphor-nucleotide moiety. Additionally, it can liberate free Co which can perpetuate metal-ion induced oxidant stress. Taken together, this is the first report of generation of toxic molecular products through the interaction of Cobl with HOCl.

We investigated the ability of reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)), hydroxyl radical ((·)OH), and hypochlorous acid (HOCl), to overcome the defensive capacity of cumulus cells and elucidate the mechanism through which ROS differentially deteriorate oocyte quality. Metaphase II mouse oocytes with (n = 1634) and without cumulus cells (n = 1633) were treated with increasing concentration of ROS, and the deterioration in oocyte quality was assessed by the changes in the microtubule morphology and chromosomal alignment. Oocyte and cumulus cell viability and cumulus cell number were assessed by indirect immunofluorescence, staining of gap junction protein, and trypan blue staining. The treated oocytes showed decreased quality as a function of increasing concentrations of ROS when compared to controls. Cumulus cells show protection against H(2)O(2) and (·)OH insult at lower concentrations, but this protection was lost at higher concentrations (>50 μmol/L). At higher H(2)O(2) concentrations, treatment dramatically influenced the cumulus cell number and viability with resulting reduction in the antioxidant capacity making the oocyte more susceptible to oxidative damage. However, cumulus cells offered no significant protection against HOCl at any concentration used. In all circumstances in which cumulus cells did not offer protection to the oocyte, both cumulus cell number and viability were decreased. Therefore, the deterioration in oocyte quality may be caused by one or more of the following: a decrease in the antioxidant machinery by the loss of cumulus cells, the lack of scavengers for specific ROS, and/or the ability of the ROS to overcome these defenses.

Interleukin 6 (IL-6) is considered a major indicator of the acute-phase inflammatory response. Endometriosis and pelvic inflammation, diseases that manifest elevated levels of IL-6, are commonly associated with higher infertility. However, the mechanistic link between elevated levels of IL-6 and poor oocyte quality is still unclear. In this work, we explored the direct role of this cytokine as a possible mediator for impaired oocyte spindle and chromosomal structure, which is a critical hurdle in the management of infertility. Metaphase-II mouse oocytes were exposed to recombinant mouse IL-6 (50, 100 and 200 ng/mL) for 30 minutes and subjected to indirect immunofluorescent staining to identify alterations in the microtubule and chromosomal alignment compared to untreated controls. The deterioration in microtubule and chromosomal alignment were evaluated utilizing both fluorescence and confocal microscopy, and were quantitated with a previously reported scoring system. Our results showed that IL-6 caused a dose-dependent deterioration in microtubule and chromosomal alignment in the treated oocytes as compared to the untreated group. Indeed, IL-6 at a concentration as low as 50 ng/mL caused deterioration in the spindle structure in 60% of the oocytes, which increased significantly (P<0.0001) as IL-6 concentration was increased. In conclusion, elevated levels of IL-6 associated with endometriosis and pelvic inflammation may reduce the fertilizing capacity of human oocyte through a mechanism that involves impairment of the microtubule and chromosomal structure.