Oxidative markers in cryopreservation medium from frozen-thawed embryos: a possible tool for improved embryo selection in in vitro fertilization?

PURPOSE: The present study evaluated the association between oxidative parameters in embryo cryopreservation medium and laboratory and clinical outcomes. METHODS: This prospective laboratory study was conducted in an IVF unit in a university-affiliated hospital with 91 IVF patients undergoing a frozen-thawed embryo transfer cycle. Following thawing, 50 µL of embryo cryopreservation medium was retrieved from each cryotube and tested by the thermochemiluminescence (TCL) assay. TCL amplitudes after 50 (H1), 150 (H2), and 280 s (H3) were recorded in counts per second (CPS) and the TCL ratio determined for comparison with implantation and pregnancy rates. RESULTS: A total of 194 embryos were transferred in 85 frozen-thaw cycles. Twenty-one pregnancies (24.7 %) occurred. Implantation and overall and clinical pregnancy rates were higher when the median TCL H1 amplitude was <32 CPS compared to ≥32 CPS (14.6 vs. 5.3 %, 37.5 vs. 17 %, 28.1 vs. 9.4 %, respectively). No pregnancies occurred when the H1 amplitude was ≥40 CPS. Logistic regression multivariate analysis found that only the median TCL H1 amplitude was associated with the occurrence of pregnancy (OR = 2.93, 95 % CI 1.065-8.08). The TCL ratio inversely correlated with the duration of embryo cryopreservation (r = -0.37). CONCLUSIONS: The results indicate that thawed embryos may express oxidative processes in the cryopreservation medium, and higher oxidative levels are associated with lower implantation rates. These findings may aid in the improved selection of frozen-thawed embryos for IVF.

Increased serum levels of oxidative stress are associated with hospital readmissions due to acute heart failure.

OBJECTIVES: Inflammation and serum oxidative stress (OS) are important components in heart failure (HF) deterioration. In this study we tested the hypothesis that an increase in patients' sera OS levels is associated with acute HF (AHF) readmissions. METHODS: Thirty consecutive patients (mean age 71 ± 10 years) admitted with AHF were included in the study. Serum OS in these patients was measured in-hospital and repeatedly after discharge over a period of 8 weeks of follow-up in which we reordered patients' HF readmissions. Of the 30 patients, 13 (43%) were readmitted (RAD group) and 17 (57%) did not require readmission (NRAD group). RESULTS: OS levels before discharge from the first hospital admission in the 2 groups were similar (p = 0.84 and p = 0.56, respectively). However, using repeated measures ANOVA, we found that the interaction between the time points and the 2 groups of patients (RAD and NRAD) was statistically significant (p = 0.037). It is important to note that OS serum levels were more predictive of HF readmissions than were repeated simultaneous serum measurements of NT-proBNP (p = 0.97). CONCLUSIONS: Increased OS levels in AHF patients, after they have been discharged from the hospital, are associated with higher HF readmission rates. In AHF, OS is a dynamic parameter associated with HF deterioration.

Serum oxidative stress level correlates with clinical parameters in chronic systolic heart failure patients.

BACKGROUND: Serum oxidative stress (OS) level has an important role in the inflammatory process of heart failure. HYPOTHESIS: The study was designed to analyze serum OS levels in chronic heart failure (HF) patients and to examine the relation between OS levels and other clinical and prognostic parameters of HF. METHODS: We studied 82 consecutive chronic symptomatic HF patients with systolic LV dysfunction (ejection fraction <45%). The serum OS level was determined using thermochemiluminescence assay. We compared the serum OS levels with patients' clinical and prognostic parameters. RESULTS: Higher serum OS levels were associated with higher New York Heart Association class (P = .01), worse renal function (serum urea, creatinine, and creatinine clearance) (P<.001) and higher serum levels of hs-C-reactive protein and N-terminal pro brain natriuretic peptide (P = .001, P<.001, respectively). CONCLUSIONS: In chronic systolic HF patients, high serum OS levels correlate with advanced disease and known markers of poor prognosis.

The effect of 100% oxygen on intestinal preservation and recovery following ischemia-reperfusion injury in rats.

OBJECTIVE: Inhalation of oxygen improves the hemodynamic status and attenuates the inflammatory response after intestinal ischemia-reperfusion (IR). Yet, the use of hyperoxia is hindered by concerns that it could exacerbate reperfusion injury by increasing free radical formation. We examined the effect of hyperoxia on enterocyte turnover and intestinal preservation and rehabilitation following IR injury in rats. DESIGN: Animal study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Animals were assigned to four experimental groups: 1) Sham rats underwent laparotomy without vascular occlusion and breathed air, 2) Sham-oxygen rats underwent laparotomy without vascular occlusion and breathed 100% oxygen, 3) IR rats underwent occlusion of the superior mesenteric artery and portal vein for 30 minutes and breathed air, and 4) IR group treated with oxygen (IR-O2)rats underwent IR and breathed 100% oxygen starting 10 minutes before and continued for the first 6 hours after reperfusion. Intestinal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined 24 hours after IR. MEASUREMENTS AND MAIN RESULTS: In IR rats, breathing 100% oxygen resulted in a significant decrease in Park's injury score in the ileum (p < 0.05 from untreated IR rats). Rats treated with oxygen also demonstrated a significant increase in mucosal weight (p < 0.05) and mucosal DNA (p < 0.05) in the jejunum and ileum, and an increase in villus height (p < 0.01), and crypt depth (p < 0.05) in the ileum. Enterocyte proliferation (assessed by bromodeoxyuridine uptake) was significantly decreased in the jejunum and ileum in untreated IR rats. Oxygen therapy increased enterocyte proliferation in the ileum, and diminished both the apoptosis index and Bax gene expression in the jejunum and ileum (p < 0.05). Plasma thermochemiluminescence oxidizability assay revealed significantly higher thermochemiluminescence ratios in IR group treated with oxygen than in untreated IR rats (p < 0.05) at 6 hours postreperfusion suggesting a significantly lower prior in vivo molecular oxidation. CONCLUSIONS: Hyperoxia reduces small bowel injury, accelerates enterocyte turnover, and improves intestinal rehabilitation after IR.

Serum oxidizability potential of ischemic heart disease patients is associated with exercise test results and disease severity.

AIM: To find out whether serum oxidizability potential correlates with exercise test (EXT) parameters and predicts their results in chronic ischemic heart disease (IHD) patients. METHODS: Oxidizability potential was determined in a group of chronic IHD patients who underwent a symptom limited EXT upon initiation of a cardiac rehabilitation program. The thermo-chemiluminescence (TCL) assay was used to assess serum oxidizability potential. This assay is based on heat-induced oxidation of serum, leading to the formation of electronically excited species in the form of unstable carbonyls, which further decompose into stable carbonyls and light energy (low chemiluminescence). Measured photons emission is represented by a kinetic curve which is described by its amplitude and slope (= ratio). We assessed the correlations of TCL ratio with exercise duration, metabolic equivalents (METS), maximal heart rate (mHR), maximal systolic BP, > 1 mm S-T depression, diabetes, hypertension, smoking, left ventricular ejection fraction (LVEF) > or < 40%, previous myocardial infarction, and aorto-coronary by-pass surgery and compared to the TCL ratio measured in a group of healthy controls. RESULTS: A high TCL ratio (%) correlated well with METS (r = 0.84), with mHR (r = 0.79) and with exercise induced S-T segment shift (r = 0.87, P < 0.05). A lower serum oxidizability potential, expressed as a low TCL ratio, thus suggestive of a previous high oxidative stress, was found in IHD patients compared to healthy controls, and, in particular, in patients with low LVEF%. The TCL ratio (%) in IHD patients was 193 ± 21, compared to 215 ± 13 in controls (P < 0.05), and was 188 ± 14.7 in patients with LVEF < 40% as compared to 200 ± 11.9 in those with LVEF > 40% (P < 0.01). A trend for lower TCL ratio (%) was found in diabetic, hypertensive, and post-coronary bypass surgery patients. A paradoxically low TCL ratio (low oxidizability potential) was observed in patients without S-T depression compared to patients with S-T depression (189 ± 22 vs 201 ± 15, P = NS), due to the fact these patients had a much lower LVEF% and a lower exercise capacity. CONCLUSION: Serum oxidizability potential is associated with EXT parameters, results, and IHD severity. TCL ratio is an "easy-to-measure marker" that might be incorporated into risk assessment and prediction in chronic IHD patients.

Oxidative stress indices in follicular fluid as measured by the thermochemiluminescence assay correlate with outcome parameters in in vitro fertilization.

OBJECTIVE: To evaluate oxidative stress indices in follicular fluid (FF) by a novel thermochemiluminescence (TCL) assay and investigate the correlation between TCL and i.v.f. cycle parameters. DESIGN: Prospective, cross-sectional study. SETTING: I.v.f. Unit of an Obstetrics and Gynecology Department in a university-affiliated hospital. PATIENT(S): One hundred eighty-nine women undergoing consecutive i.v.f. treatment cycles during 2001. INTERVENTION(S): After oocyte retrieval, pooled FF was centrifuged and the supernatant was tested in the TCL assay. MAIN OUTCOME MEASURE(S): Maximal serum E2 levels, number of gonadotropin ampoules, retrieved oocytes, mature oocytes, fertilization and cleavage rates, number of available embryos and cryopreserved embryos, and clinical pregnancy rates and correlation with TCL indices. RESULT(S): The TCL curve slope of FF positively correlated with maximal serum E2 levels, number of mature oocytes, and number of cleaved embryos and was inversely correlated with the women's ages and the number of gonadotropin ampoules. Follicular fluid TCL amplitude at 50 seconds ranged from 294 to 711 cps, but all pregnancies (n = 50; 28.1%) occurred within the range of 347-569 cps. With 385 and 569 cps as cutoff levels for the occurrence of pregnancy, the negative predictive value beyond this range was 96% and the positive predictive value within this range was 32%. CONCLUSION(S): The TCL results may reflect the age-related increase in free radical activity and is associated with parameters of ovarian responsiveness and IVF outcome. A certain threshold of oxidative stress may be required for the occurrence of conception in i.v.f. TCL is a potential tool to evaluate, treat, and monitor antioxidant therapy in i.v.f. treatments.

Oxidative stress indices in seminal plasma, as measured by the thermochemiluminescence assay, correlate with sperm parameters.

OBJECTIVE: To examine the effect of oxidation of proteins and lipids, as measured by a novel thermochemiluminescence (TCL) analyzer, and to evaluate the correlation between TCL indices in seminal plasma and sperm parameters. DESIGN: Experimental and prospective clinical studies. SETTING: An infertility unit. PATIENT(S): One hundred forty-eight men undergoing semen analysis. INTERVENTION(S): Bovine serum albumin (BSA) and linolenic acid were oxidized and tested by TCL, protein carbonyls, and conjugated dienes assays. All participants underwent semen analysis. Seminal plasma was tested by TCL and conjugated dienes. MAIN OUTCOME MEASURE(S): Thermochemiluminescence indices before and after oxidation of BSA and linolenic acid, compared with protein carbonyl and conjugated dienes indices. Correlation between semen parameters and TCL and conjugated dienes indices in seminal plasma. RESULT(S): Oxidation of BSA and linolenic acid was marked by characteristic changes in their TCL curve pattern and an increase in the levels of protein carbonyls and conjugated dienes. Among 125 sperm-containing semen samples, the TCL curve exhibited two patterns: a positive relative ratio curve (group A, 87 patients) and a negative relative ratio curve (group B, 38 patients). Sperm concentration was lower and total motile sperm and rapid motile sperm were fewer in group B. A significant correlation was found between TCL indices, conjugated dienes, and sperm quality in group B. CONCLUSION(S): Oxidation affects TCL curve pattern of proteins and lipids in a characteristic manner. Thermochemiluminescence indices in seminal plasma closely correlate with sperm characteristics among patients with sperm disturbances, and it might serve as a tool in the evaluation, treatment, and monitoring of subfertile men.

Oxidative stress in hypertensive,diabetic, and diabetic hypertensive rats.

BACKGROUND: Reactive oxygen species play a key role in the formation of endothelial dysfunction accompanying diabetes mellitus, hypertension, and other cardiovascular diseases. METHOD: This study compares oxidative stress (OS) in Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), non-insulin-dependent Cohen Diabetic rats (CDR), and Cohen Rosenthal diabetic hypertensive rats (CRDH), a unique animal model of both diabetes and hypertension. The OS was evaluated with a newly developed thermochemiluminiscence (TCL) analyzer (Lumitest Ltd., Nesher, Israel) that measures the oxidizability (ie, susceptibility to oxidation) of a test sample. RESULTS: The TCL oxidizability test results of sera from the different rats groups showed a time-dependent increase in TCL of up to 145% +/- 7% for WKY, 160% +/- 8% for SHR, 179% +/- 12% for CDR, and 226% +/- 15% for CRDH. These results were significant: P <.001 for SHR and CDR and P <.0001 for CRDH in comparison to WKY. Lipid peroxide levels also increased in each strain of rats: to 80 +/- 7.8 nmol/mL in WKY, 104 +/- 10.1 nmol/mL in SHR, 110 +/- 9.4 nmol/mL in CDR, and 167 +/- 11.7 nmol/mL in CRDH. These results were also significant: P <.001 for SHR, CDR and CRDH in comparison to WKY. CONCLUSION: The combination of hypertension and diabetes is accompanied by higher oxidative stress than that seen with either disorder alone.

Modifications and oxidation of lipids and proteins in human serum detected by thermochemiluminescence.

Detection of electronically excited species (EES) in body fluids may constitute an important diagnostic tool in various pathologies. Examples of such products are triplet excited carbonyls (TEC), which can be a source for photon emission in the 400-550 nm range. The aim of the present study was to determine the actual contribution of lipid and protein components (protein carbonyls) to photon emission generated by thermochemiluminescence (TCL) during the heating of biological fluids. In this study, a new TCL Photometer device, designed by Lumitest Ltd, Israel, was used. Samples were heated to a constant temperature of 80 +/- 0.5 degrees C for 280 s and photon emission was measured at several time points. In order to compare the results of TCL measurements to conventional methods of detecting lipid and protein oxidation, each examined sample was also heated in a waterbath at 80 degrees C for 10-280 s. Lipid and protein oxidation were subsequently measured using conventional methods. The TCL of four polyunsaturated fatty acids (PUFA) with three to six double bonds was measured. The elevation of the PUFA TCL amplitude correlated with the increase in the number of double bonds of PUFA. A correlation between the increase in TCL intensity and protein carbonyl generation in bovine serum albumin (BSA) was also observed. In the venous blood serum, our study showed that an increase of TCL intensity during heating reflected the cleavage of TEC of lipid origin. Our study suggests that biological molecules such as proteins, lipids and other molecules, which may become unstable during heating, are capable of generating EES. We demonstrated that a TCL curve can be used as a kinetic model for measuring oxidative processes, which reflects modifications of different molecules involved in the oxidative stress phenomena.