Anti-inflammatory and antioxidant effect of cerium dioxide nanoparticles immobilized on the surface of silica nanoparticles in rat experimental pneumonia.

A massage with the potent counter-inflammatory material, cerium dioxide nanoparticles, is promising and the antioxidant properties of CeO2 are considered the main, if not the only, mechanism of this action. Nevertheless, the elimination of ceria nano-particles from the organism is very slow and there is a strong concern for toxic effect of ceria due to its accumulation. To overcome this problem, we engineered a combined material in which cerium nanoparticles were immobilized on the surface of silica nanoparticles (CeO2 NP), which is shown to be easily removed from an organism and could be used as carriers for nano-ceria. In our study particle size was 220±5nm, Zeta-potential -4.5mV (in water), surface charge density -17.22µC/cm2 (at pH 7). Thirty-six male Wistar rats, 5 months old and 250-290g were divided into four groups: 1) control; 2) CeO2 NP treatment; 3) experimental pneumonia (i/p LPS injection, 1mg/kg); and 4) experimental pneumonia treated with CeO2 NP (4 times during the study in dosage of 0.6mg/kg with an orogastric catheter). Gas exchange and pulmonary ventilation were measured four times: 0, 1, 3 and 24h after LPS injection in both untreated and CeO2 NP-treated animals. The mRNA of TNF-α, Il-6, and CxCL2 were determined by RT-PCR. ROS-generation in blood plasma and lung tissue homogenates were measured by means of lucigenin- and luminol-enhanced chemiluminescence. Endotoxemia in the acute phase was associated with: (1) pathological changes in lung morphology; (2) increase of ROS generation; (3) enhanced expression of CxCL2; and (4) a gradual decrease of VO2 and VE. CeO2 NP treatment of intact animals did not make any changes in all studied parameters except for a significant augmentation of VO2 and VE. CeO2 NP treatment of rats with pneumonia created positive changes in diminishing lung tissue injury, decreasing ROS generation in blood and lung tissue and decreasing pro-inflammatory cytokine expression (TNF-α, Il-6 and CxCL2). Oxygen consumption in this group was increased compared to the LPS pneumonia group. In our study we have shown anti-inflammatory and antioxidant effects of CeO2 NP. In addition, this paper is the first to report that CeO2 NP stimulates oxygen consumption in both healthy rats, and rats with pneumonia. We propose the key in understanding the mechanisms behind the phenomena lies in the property of CeO2 NP to scavenge ROS and the influence of this potent antioxidant on mitochondrial function. The study of biodistribution and elimination of СеО2NP is the purpose of our ongoing study.

Milk fat globule epidermal growth factor 8 (MFG-E8): a novel protein in the mammalian endometrium with putative roles in implantation and placentation.

OBJECTIVES: MFG-E8 is a novel endometrial protein with conserved functions in tissue remodeling and angiogenesis in non-uterine tissues. Our aims were: 1. To examine the presence of MFG-E8 protein in the human endometrium during the window of implantation, in human endometrial cell lines, in human placental tissue at different gestational ages, and in murine implantation sites during early gestation; and 2. To study the regulation of MFG-E8 mRNA expression in mice implantation sites. STUDY DESIGN: MFG-E8 protein and its receptor integrin αvß3 were detected by immunostaining in human endometrial biopsies obtained from normal volunteers, in human endometrial cell lines (epithelial: Ishikawa and HEC-1A, stromal: HESC, and endothelial: HEEC), in human products of conception from all trimesters of gestation, and in murine implantation and inter-implantation sites dissected on days 5 and 8 post-coitus. MFG-E8 gene expression was assessed by RT-PCR. MAIN OUTCOME MEASURES: Immunohistochemical determination of MFG-E8 in endometrium and products of conception as well as relative MFG-E8 mRNA expression in mice implantation sites. RESULTS: MFG-E8 protein was present almost exclusively in the epithelial compartment of human endometrium. It was also expressed in the cytotrophoblasts and syncytiotrophoblasts outlining chorionic villi of the human placenta at all trimesters of gestation, and in murine implantation sites. MFG-E8 mRNA was significantly up-regulated in murine implantation sites and with increased gestational age. CONCLUSIONS: MFG-E8 expression in the endometrial epithelium as well as in chorionic villi suggests its possible role in endometrial reorganization during the receptive phase and in events related to normal pregnancy in mammals.

Intermittent hypoxia: mechanisms of action and some applications to bronchial asthma treatment.

Being essentially cut off from the global scientific community, Ukrainian and Russian scientists have developed a new concept for the beneficial use of adaptation to artificial intermittent hypoxia in treating of many diseases. The basic mechanisms underlying intermittent hypoxic training were elaborated mainly in three areas: regulation of respiration, free radical production and mitochondrial respiration. Twenty-year experience of the application of intermittent hypoxic therapy for the treatment of chronic obstructive bronchitis and bronchial asthma allows affirming that the adaptation to this kind of hypoxia causes a significant improvement of the clinical picture or even a complete recovery. The absence of negative side effects, typically observed during drug therapy, and the stimulation of organism's general, nonspecific resistance, makes the hypoxic therapy a treatment with a future. A special note is devoted to the use of intermittent hypoxic training in industrial health care for the purpose of prophylaxis and treatment of professional diseases.

Geriatric men at altitude: hypoxic ventilatory sensitivity and blood dopamine changes.

BACKGROUND: Short-term exposure to high-altitude hypoxia increases hypoxic ventilatory sensitivity (HVS) in healthy humans. Dopamine (DA) is the implicated neurotransmitter in carotid body (CB) chemoreceptor response, and the microenvironmental conditions in CB tissue are comparable to blood. Continuous DA infusion affected ventilation in animals and humans. Age-related oscillations in blood DA levels may influence peripheral chemoreflexes. OBJECTIVE: Hypoxic ventilatory responses (HVR) relative to blood DA concentration and its precursor, dihydroxyphenylalanine (DOPA) was measured in young and elderly men during short-term altitude adaptation. METHODS: Nine elderly climbers (group 1:61+/-1.4 years) and 7 young healthy subjects (group 2: 23+/-2 years) were tested at sea level on day 0, on day 3 after passive transport to 2,200 m, and on day 14 after climbing to 4,200 and 5,642 m. RESULTS: Sea level HVR in group 1 was 47% lower than in group 2, accompanied by higher blood DOPA (300%) and DA (37%) content. Initial DA and DOPA concentrations showed a negative correlation with initial HVR but a positive correlation with age. Passive transport to middle altitude (2,200 m) increased HVS, doubling HVR slopes in groups 1 and 2 and producing increased maximum expired minute ventilation during isocapnic rebreathing (29 and 28%, respectively). Day 3 2,200-meter blood DOPA content decreased by 22% in group 1 and increased by 300% in group 2. DA increased in both groups. CONCLUSION: The relationship between HVR and the reciprocal DA and DOPA values seen in both groups is associated with age, producing decreased DA receptor sensitivity and enhanced DA reuptake during adaptation to high altitude.

Mechanisms of taurine antihypoxic and antioxidant action.

The study was undertaken to elucidate the effects of taurine on lipid peroxidation (LP) intensity and membrane Na+, K+-ATPase activity in a hypoxic rat model. It was shown that 3 intraperitoneal (i.p.) injections of 200 mg/kg of taurine prevented hypoxia-induced lactate accumulation and LP in brain, liver, and heart tissues and prevented the decrease of Na+, K+-ATPase activity in the liver. It is suggested that the effect of taurine on LP could be due to the taurine antiacidotic action as well as to its membrane stabilizing activity.

Human hypoxic ventilatory response with blood dopamine content under intermittent hypoxic training.

Adaptation to intermittent hypoxia can enhance a hypoxic ventilatory response (HVR) in healthy humans. Naturally occurring oscillations in blood dopamine (DA) level may modulate these responses. We have measured ventilatory response to hypoxia relative to blood DA concentration and its precursor DOPA before and after a 2-week course of intermittent hypoxic training (IHT). Eighteen healthy male subjects (mean 22.8+/-2.1 years old) participated in the study. HVRs to isocapnic, progressive, hypoxic rebreathing were recorded and analyzed using piecewise linear approximation. Rebreathing lasted for 5-6 min until inspired O2 reached 8 to 7%. IHT consisted of three identical daily rebreathing sessions separated by 5-min breaks for 14 consecutive days. Before and after the 2-week course of IHT, blood was sampled from the antecubital vein to measure DA and DOPA content. The investigation associated pretraining high blood DA and DOPA values with low HVR (r = -0.66 and -0.75, respectively), elevated tidal volume (r = 0.58 and 0.37) and vital capacity (r = 0.69 and 0.58), and reduced respiratory frequency (r = -0.89 and -0.82). IHT produced no significant change in ventilatory responses to mild hypoxic challenge (Peto2 from 110 to 70-80 mm Hg; 1 mm Hg = 133.3 Pa) but elicited a 96% increase in ventilatory response to severe hypoxia (from 70-80 to 45 mm Hg). Changes in HVRs were not accompanied by statistically significant shifts in blood DA content (24% change), although a twofold increase in DOPA concentration was observed. Individual subject's changes in DA and DOPA content were not correlated with HVR changes when these two parameters were evaluated in relation to the IHT. We hypothesize that DA flowing to the carotid body through the blood may provoke DA autoreceptor-mediated inhibition of endogenous DA synthesis-release, as shown in our baseline data.

Intermittent hypoxia alters hypoxic ventilatory responses.

Intermittent hypoxic training (IHT) shows promise for prevention and treatment of some diseases and efficiently produces great advancement in athletic training. We studied (1) hypoxic ventilatory responses (HVR) in supine and sitting positions during normobaric, isocapnic, progressive hypoxia (rebreathing technique) and (2) lung ventilation and gas exchange while breathing ambient air at rest and during 5 min of breathing 11% O2. Duel measurements were made pre- and post-15-day IHT regimen on 12 (experimental) healthy males (24.6 y.o. +/- 1.9 y.o.) and on 6 (control) healthy males (24.2 y.o. +/- 2.3 y.o.) given pseudo-IHT (p-IHT) without decreasing PiO2. IHT involved rebreathing eucapnic (chemically absorbed) air as P(ET)O2 decreased to 35 mmHg, three 6-7 min sessions, three times a day, with 10 min breaks between each session over a 15 day training period. Without IHT, HVRs were the same in sitting and supine positions at low levels of hypoxic challenge (slope one--S1: P(ET)O2 from 110-60 mm Hg) and significantly higher (by 45%) during severe hypoxia (slope two--S2: P(ET)O2 from 60-35 mm Hg). IHT caused an increase in HVR in both sitting and supine positions: S1 by 70 and 100%, S2 by 158 and 200%, maximal lung ventilation by 35 and 78%, respectively. There were no significant changes in the p-IHT group. IHT also caused enhanced respiratory reactions during sustained hypoxia (lung and alveolar ventilation increased by 36 and 22%, respectively). A striking hypoxic ventilatory sensitivity was noted in subjects with hyper-reactive breathing patterns.

Value of sperm morphology assessed by strict criteria for prediction of the outcome of artificial (intrauterine) insemination.

The purpose of this study was to investigate the relationship between sperm parameters and the outcome of artificial (intrauterine) insemination (IUI). One hundred and twenty-six patients undergoing 395 consecutive IUI cycles in a 1-year period in our institution were studied. In all cases, controlled ovarian stimulation and hCG-timed IUI were performed, followed by progesterone supplementation of the luteal phase. In 86 patients, (243 cycles) the husband's sperm was utilized, whereas in 40 couples (152 cycles), donor sperm was used due to severe male factor infertility. Among the sperm parameters of the original ejaculate, % normal morphology (assessed by strict criteria) was the most significant predictor of pregnancy (stepwise regression analysis, P = 0.003). Using logistic regression, morphology was the best predictor of pregnancy (r = 0.12); linearity of movement significantly enhanced the predictive value of morphology alone (r = 0.17, P = 0.004). Overall, the pregnancy rates were significantly higher (P = 0.01) in cases with > or = 14% normal morphology (15% per cycle) compared to cases with < 14% normal morphology (7% per cycle). This threshold level for morphology is in agreement with previously published IVF results. These results underscore the significance of sperm morphology assessed by strict criteria as a predictor of pregnancy outcome in the IUI setting.

Sperm morphology as diagnosed by strict criteria: probing the impact of teratozoospermia on fertilization rate and pregnancy outcome in a large in vitro fertilization population.

OBJECTIVE: To investigate the predictive value of sperm morphology assessed by strict criteria on IVF outcome. DESIGN: Retrospective analysis of all IVF cycles (January 1987 to December 1992). MAIN OUTCOME MEASURES: All patients were assigned to one of three groups based on sperm morphology: P-pattern (< 4% normal forms), G-pattern (4% to 14% normal forms), and N-pattern (> 14% normal forms). Morphology pattern was related to other semen characteristics and IVF outcome. RESULTS: Despite corrective measures at oocyte insemination, the fertilization rate was significantly different among the three morphology groups, P < G < N. N-pattern sperm produced a mean fertilization rate over 85% regardless of low motility or concentration. In a cohort study, P-pattern cycles produced a lower implantation rate and lower ongoing pregnancy rate, independent of the lower fertilization rate. CONCLUSIONS: Strict morphology is an excellent biomarker of sperm fertilizing capacity, independent of motility and concentration. P-pattern sperm may denote a poorer prognosis for establishing a pregnancy, even after a satisfactory fertilization rate is achieved.

Enhancement of in vitro murine embryo development by recombinant leukemia inhibitory factor.

OBJECTIVE: Human recombinant leukemia inhibitory factor (rLIF) has been shown to stimulate hatching of murine and ovine embryos in vitro. The temporal and dose-dependent effects of murine rLIF (mrLIF) and human rLIF (hrLIF) on embryo development in two different mouse strains were investigated in this work. METHODS: Two-cell embryos were recovered from the fallopian tubes of superovulated/mated females and cultured in Krebs medium plus bovine serum albumin in microdroplets under oil. RESULTS: In the B6CBF1 strain, mrLIF significantly stimulated blastocyst formation and decreased embryo fragmentation/degeneration when added simultaneously at the initiation of culture or 24 hours thereafter. Human rLIF also had a positive effect on development. In the CD1 strain (lower fecundity), mrLIF dose-dependent effects were observed, with enhanced developmental stimulation achieved with higher doses. CONCLUSIONS: These findings confirm that hrLIF stimulates mouse embryo development in vitro and that different mouse strains show distinct responses to the cytokine. In addition, mrLIF enhances blastocyst formation and decreases embryo fragmentation when added to the embryo culture as early as the two-cell stage.

The YAG laser used in micromanipulation to transect the zona pellucida of hamster oocytes.

PROBLEM: Since there has been no reported use of the YAG laser to micromanipulate oocytes, our purpose was to study whether (1) a YAG laser could be used to open the zona pellucida of hamster oocytes; (2) human sperm could reach the ooplasm and (3) under sperm penetration assay conditions, sperm would bind and penetrate the ooplasm. RESULTS: A YAG 100 laser was used at 10 W and 0.4-sec pulse width to open eight of eight ooplasm oocytes. The opening in the zonae was 0.25 to 1.0 rad (10 to 40 microns). For the initial eight oocytes and two parallel controls, the coarse appearance of the ooplasm was unchanged after 3 days. Next, in 11 of 12 manipulated oocytes, the sperm clustered at the opening of the zona. When 16 more oocytes were opened and exposed to sperm in sperm penetration assay conditions, each ooplasm bound sperm. There was no penetration noted. Each manipulation time was < 1 min. To clarify the laser effect, oocytes were exposed to laser energy then utilized as the interactive surface in the sperm penetration assay. It was found that only 20% bound sperm with no penetration. CONCLUSION: While the time factor compares favourably with other methods of zona opening, further study needs to be performed to minimize effect to the exposed oocyte.

Fertilization and mouse embryo development in the presence of midazolam.

Mouse embryo in vitro development elucidates the effect of a pharmacologic agent on cellular differentiation. Midazolam provides conscious sedation for patients undergoing egg retrieval for in vitro fertilization and is found in patient follicular fluid. Mouse preimplantation embryo formation and development were evaluated in the presence of midazolam. Midazolam was cocultured with two-cell mouse preimplantation embryos over 72 h and injected systemically just before ovulation and coitus. Concentrations to 12.5 micrograms/mL displayed no significant toxic effects on in vitro two-cell-to-blastocyst development. Doses to 35.0 mg/kg did not prevent or impair in vivo fertilization. Midazolam has no adverse effect on in vitro development of two-cell-to-blastocyst-stage embryos nor on in vivo fertilization and cell division at concentrations approximating and exceeding those that ova are exposed to during clinical anesthesia. Midazolam is recommended for use to induce sedation in human in vitro fertilization where association with gametes and zygotes is probable.

Relationship between morphology and motion characteristics of human spermatozoa in semen and in the swim-up sperm fractions.

In this study, the authors evaluated the morphology pattern and motion characteristics of human spermatozoa before and after swim-up separation. Samples were divided into two, morphologically different groups according to the percentage of normal sperm forms assessed by the strict criteria of the Norfolk laboratory: "good"(G) and "poor" (P) prognosis patterns. The percentages of normal forms, slightly abnormal forms, and severe head defects were significantly different in the two groups. Motile characteristics were analyzed by a computerized semen analyzer with constant parameter settings. Before swim-up there were no significant differences in semen volume, percentage of neck and tail defects, concentration, or percentage of motility and linearity, but the mean velocity was higher in group G. After swim-up the percentage of motility, total number of motile cells, and recovery rate were higher for group G, and the incidence of severe head defects correlated negatively with the percentage of cells with a velocity of greater than 80 microns/sec. The results suggest that patients with a high incidence of sperm head defects have impaired original velocity, and swim-up selects for velocity as well as normal forms and motility. Although motility and velocity improved substantially after swim-up, the recovery rate and percentage of motility were significantly lower in the P group.

Comparison of a discontinuous Percoll gradient method versus a swim-up method: effects on sperm morphology and other semen parameters.

Increasing the proportion of motile and normal spermatozoa is an important objective for assisted reproductive procedures. The aim of this study was to compare the results of a Percoll and swim-up-method, especially on the sperm morphology. The Percoll technique gave a recovery rate of 81.3% of motile spermatozoa against a recovery rate of 28.98% for the swim-up technique. When the percentage of normal spermatozoa was also brought into calculation the recovery rate with the Percoll technique decreased to 62.03% and increased to 31.81% for the swim-up technique. This was due to a decrease in the resultant morphology of the Percoll technique to a median of 8.0% and an increase to 11.5% for the swim-up technique from a median of 10.5% of the original samples.

Hypoosmotic swelling test in the prediction of male fertility.

The hypoosmotic swelling test (HOS) is a simple test to measure the functional competence of human sperm membranes. The question is, does a relationship exist between this competence and the fertilization potential of human spermatozoa? In this study the strongest correlation (r = 0.76) was obtained between sperm swelling and sperm viability (supravital staining). Only a moderate correlation (r = 0.50) was obtained with normal sperm morphology; weaker correlations were also obtained with the sperm penetration assay (r = 0.42) and human IVF (r = 0.24). The results, therefore, indicate that the HOS test has a limited predictive value. Notwithstanding this low concordance between sperm swelling and fertilizing potential, a less than 50% HOS test threshold was seen to be a definite indicator of a male factor.

Usefulness of sperm penetration assay in fertility predictions.

The competence of the sperm penetration assay (SPA) to predict male fertility, as determined by normal sperm morphology and the fertilizing potential, as shown by human in vitro fertilization (IVF), was investigated. A significant correlation was obtained between normal sperm morphology and the SPA (phi = 0.623). A weaker correlation was however obtained with human IVF (phi = 0.397). Notwithstanding this weak association, a positive SPA (greater than 10%) was highly predictive (95%) of human IVF success. In contrast, a negative SPA (less than or equal to 10%) was associated with a high rate of false-negatives (65%). The SPA does however warn that a male factor may be present, as the mean fertilization rate of this group of patients was markedly reduced. The preincubation period for the spermatozoa did not play a major role in the predictive ability of a SPA outcome.

Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities.

Sperm morphology evaluated by new, strict criteria is a good predictor of outcome in in vitro fertilization (IVF). This study aimed (1) to determine whether the fertilization rate of preovulatory oocytes in patients with abnormal morphology can be improved by increasing insemination concentration at the time of IVF and (2) to evaluate the pregnancy outcome in patients with abnormal sperm morphology. Three groups were studied: (1) normal morphology, (2) good prognosis pattern, and (3) poor prognosis pattern. All other sperm parameters were normal. Group 3 had a lower overall fertilization rate, lower pregnancy rate/cycle, and lower ongoing pregnancy rate/cycle. Groups 2 and 3 showed a higher miscarriage rate, although not significantly different from group 1. By increasing insemination concentration from 2- to 10-fold, the fertilization rate in group 3 increased from 14.5% to 62.6%. However, pregnancy outcome did not improve. We conclude that patients with severe sperm head abnormalities have a lower ability to establish successful pregnancies, even though fertilization may be achieved.

Mouse embryo quality control for toxicity determination in the Norfolk in vitro fertilization program.

Ongoing quality control is necessary as part of the maintenance and improvement of a successful human in vitro fertilization (IVF) program. Using a mouse quality-control culture system, several instrument preparation protocols were reevaluated to determine their efficiency in the control or elimination of potential toxicity. Dilute concentrations of urine and endometrial fluid were also tested. Medium rinsed through laparoscope and aspiration needles failed to support embryo development. This effect was reversed in needles that were pretreated with rinses of Dulbecco's phosphate-buffered saline. Endometrial fluid demonstrated no obvious toxic effect, but urine-exposed embryos arrested in the two-cell state. The importance of periodic evaluation of materials and their pretreatment before use in in vitro fertilization of human oocytes is essential to ensure control of potentially toxic substances.

Evaluation of semen parameters by means of automated sperm motion analyzers.

Fresh semen specimens from 46 patients and donors were evaluated for concentration, motility, velocity, and linearity using two different commercially available computerized sperm motion analyzer systems. Although no significant differences in measurement of concentration or motility were observed, significant differences in velocity and linearity were recorded. Fourteen cryopreserved/thawed samples were assessed with the same set-up parameters as fresh specimens. When discrepancies between manual and computer counts were noted, the authors changed the set-up parameters and evaluated 33 additional specimens. Again, no differences in concentration and motility, but significant differences in velocity and linearity were observed. Interlaboratory results must be correlated and standardization of set-up parameters of various analyzers is essential.