Cyclophosphamide and acrolein induced oxidative stress leading to deterioration of metaphase II mouse oocyte quality.

Cyclophosphamide (CTX) is a chemotherapeutic agent widely used to treat ovarian, breast, and hematological cancers as well as autoimmune disorders. Such chemotherapy is associated with reproductive failure and premature ovarian insufficiency. The mechanism by which CTX and/or its main metabolite, acrolein, affect female fertility remains unclear, but it is thought to be caused by an overproduction of reactive oxygen species (ROS). Here, we investigated the effect of CTX on metaphase II mouse oocytes obtained from treated animals (120mg/kg, 24h of single treatment), and oocytes directly exposed to increasing concentrations of CTX and acrolein (n=480; 0, 5, 10, 25, 50, and 100µM) with and without cumulus cells (CCs) for 45min which correlates to the time of maximum peak plasma concentrations after administration. Oocytes were fixed and subjected to indirect immunofluorescence and were scored based on microtubule spindle structure (MT) and chromosomal alignment (CH). Generation of ROS was evaluated using the Cellular Reactive Oxygen Species Detection Assay Kit. Deterioration of oocyte quality was noted when oocytes were obtained from CTX treated mice along with CTX and acrolein treated oocytes in a dose-dependent manner as shown by an increase in poor scores. Acrolein had an impact at a significantly lower level as compared to CTX, plateau at 10µM versus 50µM, respectively. These variation is are associated with the higher amount of ROS generated with acrolein exposure as compared to CTX (p<0.05). Utilization of antioxidant therapy and acrolein scavengers may mitigate the damaging effects of these compounds and help women undergoing such treatment.

Diffused Intra-Oocyte Hydrogen Peroxide Activates Myeloperoxidase and Deteriorates Oocyte Quality.

Hydrogen peroxide (H2O2) is a relatively long-lived signaling molecule that plays an essential role in oocyte maturation, implantation, as well as early embryonic development. Exposure to relatively high levels of H2O2 functions efficiently to accelerate oocyte aging and deteriorate oocyte quality. However, little precise information exists regarding intra-oocyte H2O2 concentrations, and its diffusion to the oocyte milieu. In this work, we utilized an L-shaped amperometric integrated H2O2-selective probe to directly and quantitatively measure the real-time intra-oocyte H2O2 concentration. This investigation provides an exact measurement of H2O2 in situ by reducing the possible loss of H2O2 caused by diffusion or reactivity with other biological systems. This experiment suggests that the intra-oocyte H2O2 levels of oocytes obtained from young animals are reasonably high and remained constant during the procedure measurements. However, the intra-oocyte H2O2 concentration dropped significantly (40-50% reduction) in response to catalase pre-incubation, suggesting that the measurements are truly H2O2 based. To further confirm the extracellular diffusion of H2O2, oocytes were incubated with myeloperoxidase (MPO), and the diffused H2O2 triggered MPO chlorinating activity. Our results show that the generated hypochlorous acid (HOCl) facilitated the deterioration in oocyte quality, a process that could be prevented by pre-incubating the oocytes with melatonin, which was experimentally proven to be oxidized utilizing HPLC methods. This study is the first to demonstrate direct quantitative measurement of intracellular H2O2, and its extracellular diffusion and activation of MPO as well as its impact on oocyte quality. These results may help in designing more accurate treatment plans in assisted reproduction under inflammatory conditions.

Melatonin prevents myeloperoxidase heme destruction and the generation of free iron mediated by self-generated hypochlorous acid.

Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl-mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

Direct real-time measurement of intra-oocyte nitric oxide concentration in vivo.

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70-75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process.

Altered endometrial expression of endothelial nitric oxide synthase in women with unexplained recurrent miscarriage and infertility.

Endothelial nitric oxide synthase (eNOS) has diverse roles in the female reproductive system including a role in blastocyst implantation. Aberrant expression of eNOS could therefore be significant in the pathogenesis of disorders of implantation. In this study, eNOS protein and mRNA levels in the endometrium of women with recurrent miscarriages, unexplained infertility and a control group were determined by compartmental quantitative immunohistochemistry and real-time reverse-transcription PCR. eNOS was found to be immunolocalized to all layers of the endometrium and vascular endothelium. eNOS protein was higher in glandular epithelium (P = 0.004) and luminal epithelium (P = 0.002), but not vascular endothelium and stroma, in women with recurrent miscarriage. Similarly, in women with unexplained infertility, eNOS was significantly higher (P < 0.03) in luminal epithelium but not in any other compartments compared with the control group. The levels of mRNA confirmed the protein data, demonstrating higher eNOS mRNA in the endometrium of women with recurrent miscarriage and unexplained infertility compared with controls. In conclusion, increased expression of eNOS in glandular and luminal epithelium of the endometrium in women with recurrent miscarriages and unexplained infertility suggests a detrimental effect of excess nitric oxide in endometrial receptivity and implantation.

Immunohistochemical localization of endothelial nitric oxide synthase in endometrial tissue of women with unexplained infertility.

BACKGROUND: Nitric oxide (NO) is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies suggested the possible role of endothelial isoform of nitric oxide synthase (eNOS) enzyme in female infertility. OBJECTIVE: The aim of this study is to evaluate the expression of endothelial nitric oxide synthase in endometrial tissue of women with unexplained infertility. MATERIALS AND METHODS: In this case-control study a total of 18 endometrial tissues obtained from 10 women with unexplained infertility and 8 normal and fertile women by endometrial biopsy, 6 to 10 days after LH surge. Specimens were fixed in 4% paraformaldhyde fixative and frozen sectioned for semi-quantitative immunohistochemical evaluation using monoclonal anti-human eNOS antibody. Hematoxilin and Eosin was used for Histological dating. RESULTS: Localization of endothelial nitric oxide synthase was seen in glandular and luminal epithelium, vascular endothelium and stroma in both fertile women and women with unexplained infertility. Although there were differences in immunoreactivity of glandular epithelium (p=0.44), vascular endothelium (p=0.60) and stroma (p=0.63) but only over-expression of eNOS in luminal epithelium (p=0.045) of women with unexplained infertility compared to fertile women was statistically significant (p<0.05). CONCLUSION: This study suggests that changes in luminal expression of eNOS may influence receptivity of endometrium.

Immuno-histochemical localization of endothelial nitric oxide synthase in testicular cells of men with non-obstructive azoospermia.

BACKGROUND: Non obstructive azoospermia (NOA) is one of the causes of male infertility in which spermatogenesis process is disturbed. Recent studies suggested the possible role of endothelial nitric oxide synthase (eNOS) in spermatogenesis process. OBJECTIVE: The aim of the present study is to evaluate the expression of eNOS in human testicular tissue in men with NOA and men with normal spermatogenesis by using immunocytochemistry. MATERIALS AND METHODS: In this case-control study, testicular biopsies were obtained from 10 men with NOA and 7 men with normospermia who were attended to infertility center for diagnosis or infertility treatment. Immunohistochemistry was used to localize the isoform of eNOS in these tissues and the intensity of staining was semi quantitively assessed. In addition, the histopathological evaluation was examined in both groups. RESULTS: The isoform of eNOS enzyme activity was detected in the cytoplasm of sertoli and leydig cells in both groups. There was, however, a considerable variability in the intensity of staining between two groups. The expression of eNOS in Leydig cells in control group was significantly (p<0.05) higher than those in the NOA group. In contrast, expression of eNOS in Sertoli cells in NOA was more than those in the control group. eNO Simmune staining was absent in the normal germ cells but was intense in the abnormal germ cells with piknotic neucleous. The most histopathological finding were hypospermatogenesis (27.2%), Sertoli cell only syndrome (18.1%) and tubular fibrotic (13.6%). CONCLUSION: These results suggested that increase level of eNOS may play an important role in the apoptosis process in the abnormal germ cells and disturbance of spermatogenesis process.

Increase in concentration of soluble HLA-G in high-quality embryos after intracytoplasmic sperm injection.

Non-invasive methods are normally preferred to conventional invasive methods when selecting suitable embryos to improve pregnancy rates after assisted reproduction techniques. One of the most recognized non-invasive methods is to examine the supernatants of embryo culture media. Soluble human leukocyte antigen, class I, G (sHLA-G) antigen is a non-classical class I molecule that has been widely considered as a marker of pregnancy failure or implantation success. In the current study of some Iranian patients, we examined the concentration of sHLA-G at different time points after intracytoplasmic sperm injection and compared the rates to the morphology and quality of the selected embryos. We showed that the concentration of sHLA-G increases over time in high-quality embryos. We conclude that there is a positive relationship between morphology, quality, and sHLA-G concentration. We suggest that this relationship can be used to increase the chance of a successful pregnancy.