Critical evaluation of two models of flow cytometers for the assessment of sperm DNA fragmentation: An appeal for performance verification / 亚洲男科学杂志(英文版)

Lack of standardized, reproducible protocols and reference values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation (SDF) is a unique example. Any change in the setting of the standard instrument, including upgrades of hardware or software, can lead to different results and may affect clinicians' decision for treatment. Therefore, we compared TUNEL results of SDF obtained from a standard (C6) flow cytometer with a newer version of the same instrument (C6 Plus) and examined the cutoff, sensitivity, and specificity without calibration (adjustment) and after adjustment. Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers. The strength of agreement of the results between the two observers was also assessed. After adjustment of the settings, overall concordance became high and the two cytometers showed 100% positive and negative predictive value with 100% area under the curve. The overall correlation coefficient observed between C6 and C6 Plus was highly significant (P 0.0001; r = 0.992; 95% confidence interval [CI]: 0.982-0.997). After adjustment, the two cytometers showed very high precision of 98% and accuracy of 99%. The interobserver agreement on C6 flow cytometer for the two observers was 0.801 0.062 and 0.746 0.044 for C6 Plus. We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.

Hippocampal neuronal apoptosis in rat offspring due to gestational diabetes / Apoptosis neuronal del hipocampo en crías de ratas debido a la diabetes gestacional

Gestational diabetes mellitus (GDM) defined as impaired glucose tolerance affects approximately 6 % of all pregnant women who have never before had diabetes, but who do have high blood glucose levels during pregnancy. This study was done to evaluate the apoptosis in the neuronal cells in the CA1, CA2 and CA3 subfields of hippocampus and dentate gyrus in offspring of gestational diabetes at the 7, 21 and 28 d in postnatal rats. Thirty Wistar rat dams were randomly allocated in control and diabetic group. Dams in diabetic group were received 40 mg/kg/BW of streptozotocin at the first day of gestation and control groups received an equivalent volume normal saline injection intraperitoneally (IP). Six offspring of GDM and control dams, at the 7, 21, 28 postnatal day were randomly were sacrificed quickly with anesthesia. The coronal sections of brain serially collected. The apoptosis neurons were evaluated with TUNEL Assay. In the CA1, the number of apoptotic cells in 7, 21 and 28 d of postnatal life were significantly increased in GDM compared to controls (P<0.001). In the CA2, CA3 the number of apoptotic cells in 7, 21 and 28 d age-old offspring were significantly increased in GDM compared to controls (P<0.001). In the dentate gyrus, the number of apoptotic cells in 7, 21 and 28 d of postnatal life were significantly increased in GDM compared to controls (P<0.01). This study showed that the uncontrolled gestational diabetes significantly increases neuronal apoptosis in hippocampal and dentate gyrus in rat offspring.

La diabetes mellitus gestacional (DMG) se define como la intolerancia a la glucosa que afecta aproximadamente al 6 % de todas las mujeres embarazadas que nunca han tenido diabetes, pero que sí tienen niveles de glucosa en la sangre elevados durante el embarazo. El objetivo de este estudio fue evaluar la apoptosis de células neuronales en CA1, CA2 y CA3, subcampos del hipocampo y el giro dentado, en las crías de ratas con diabetes gestacional en los días 7, 21 y 28 luego del nacimiento. Se utilizaron 30 ratas Wistar asignadas aleatoriamente en grupos control y diabético (GDM). Se administró al grupo diabético 40 mg/kg de peso corporal de estreptozotocina en el primer día de gestación y el grupo control recibió un volumen equivalente de solución salina normal por inyección vía intraperitoneal. Seis crías de los grupos GDM y control fueron seleccionadas aleatoriamente y sacrificadas bajo anestesia los días 7, 21, 28. Se tomaron secciones seriales coronales del cerebro. La apoptosis neuronal se evaluó mediante ensayo TUNEL. En el CA1, el número de células apoptóticas a los 7, 21 y 28 d se incrementó significativamente en el grupo GDM en comparación con los controles (P <0.001). En el CA2 y CA3 el número de células apoptóticas en los días 7, 21 y 28 también se incrementó significativamente en GDM en comparación con los controles (P <0,001). En el giro dentado, el número de células apoptóticas en los días 7, 21 y 28 se incrementó significativamente en GDM en comparación con los controles (P <0,01). Este estudio mostró que la diabetes gestacional no controlada aumenta significativamente la apoptosis neuronal en el hipocampo y el giro dentado en las crías de las ratas.

Ganglionic cells apoptosis in retinal layer of rat offspring due to gestational diabetes / Células ganglionares apoptóticas en la capa de la retina de crías de ratas debido a la diabetes gestacional

Previous studies have shown the adverse effects of gestational diabetes on hippocampal neuronal density in animal models. This study was conducted to determine the effect of gestational diabetes on retinal ganglionic cell density, the thickness of the retinal layer and apoptotic ganglionic cell density in 28-day-old of rat offspring. In this experimental study, 10 Wistar rat dams were randomly allocated in control and diabetic groups. Gestational diabetes was induced by 40 mg/kg/body weight of streptozotocin at the first day of gestation, intraperitoneally, dams in control group received an equivalent volume normal saline. At postnatal day 28, six offspring of each gestational diabetes and controls were randomly selected, sacrificed and sections (6 micrometer) were taken from the eye and stained with hematoxylin and eosin. The density of ganglionic cells and the number of dUTP end-labeling (TUNEL)-positive cells were evaluated in 20000 mm2 area of ganglion layer of the retina. The ganglionic cells density were reduced (27.4%) in gestational diabetic offspring in compared to controls (22.5±1.5 vs. 31.0±0.9, P<0.01). The apoptotic ganglionic cells of retina in interventional group significantly increased in compared to controls (6.74±0.60 vs. 5.12±0.26, P<0.02). This study showed that the uncontrolled gestational diabetes can reduce the number of ganglionic cells and increase apoptotic ganglionic cells of retina layer in rat offspring.

Estudios previos en un modelo animal han demostrado los efectos adversos de la diabetes gestacional en la densidad neuronal del hipocampo. El objetivo fue determinar el efecto de la diabetes gestacional en la densidad de las células ganglionares de la retina, en el espesor de la capa de la retina y en la densidad de las células apoptóticas ganglionares, en crías de ratas de 28 días. En este estudio experimental, 10 ratas Wistar fueron asignadas aleatoriamente en grupos control y diabéticos. La diabetes gestacional se indujo a partir de la administración de 40 mg/kg/peso corporal de estreptozotocina en el primer día de la gestación, por vía intraperitoneal. Al grupo control se administró un volumen equivalente de solución salina normal. En el día 28 luego del nacimiento, se seleccionaron aleatoriamente seis crías procedentes de los grupos con diabetes gestacional y controles, se eutanasiaron y se tomaron muestras de los ojos, en forma de secciones de 6 micrómetros, las cuales se tiñeron con H & E. La densidad de las células ganglionares y el número final de células dUTP positivas (TUNEL) se evaluaron a nivel de la capa ganglionar de la retina, en un área de 20.000 mm2. La densidad de las células ganglionares se redujo un 27,4% en la descendencia con diabetes gestacional en comparación con los controles (22,5±1,5 vs. 31,0±0,9, P<0,01). Las células ganglionares apoptóticas de la retina en el grupo con diabetes gestacional aumentaron significativamente en comparación con los controles (6,74±0,60 vs. 5,12 ± 0,26, P <0,02). Este estudio demostró que la diabetes gestacional no controlada puede reducir el número de células ganglionares y aumentar el número de células ganglionares apoptóticas de la capa de la retina en las crías de las ratas con diabetes gestacional.

Preventing effect of vitamin E on oocytes apoptosis in morphine-treated mice / Efecto preventivo de la vitamina E sobre la apoptosis de ovocitos en ratones tratados con morfina

Several studies have shown that Morphine Sulfate affects on fertility, embryogenesis and consequent pregnancy loss and ultrastructural alterations of oocytes in animal model. This study was done to determine the effect of morphine sulfate on oocytes apoptosis and preventive role of daily supplementation of Vitamin E on oocytes apoptosis in morphine sulfate -treated mice. Twenty-four NMARI female mice were randomly allocated into four experimental groups. For 15 days, control group received saline (0.2 ml/day by subcutaneous injection), group I Vitamin E (60 mg/kg/day orally), group II Morphine Sulfate (10 mg/kg/day by subcutaneous injection) and group III Morphine Sulfate with Vitamin E (60 mg/kg/day orally). Then, animals were superovulated with PSMG (10 Units) and 10 Unites of HCG. The next day the animals were sacrificed, oocytes were flushed from each fallopian tube. The collected oocytes were subjected to determine apoptosis by Tunnel assay with using Fluorescent Microscope. According to our results, the number of retrieved oocytes were 121, 132, 86 and 114 in control, experimental group I, II and III, respectively. Morphine Sulfate treatment increased apoptosis in oocytes to 17.44 percent whereas oocytes apoptosis was 4.13 percent in Controls. Supplementation with Vitamin E in Morphine Sulfate -treated mice reduced the oocytes apoptosis to 7.01 percent. This study showed that Morphine can increase apoptosis in oocytes and Vitamin E treatment significantly reduces oocytes apoptosis in the Morphine Sulfate -treated mice.

Diversos estudios han demostrado que el sulfato de morfina afecta la fertilidad, embriogénesis y en consecuencia pérdida de la preñez y alteraciones ultraestructurales de los ovocitos en el modelo animal. Este estudio determinó el efecto del sulfato de morfina sobre la apoptosis de los ovocitos y papel preventivo de la suplementación diaria de la vitamina E en la apoptosis de ovocitos en ratones tratados con sulfato de morfina. Veinte y cuatro ratones NMARI hembras fueron asignados al azar en 4 grupos experimentales. Durante 15 días, el grupo control recibió solución salina (0,2 ml/día por inyección subcutánea), el grupo I vitamina E (60 mg/kg/día por vía oral), el grupo II Sulfato de morfina (10 mg/kg/día por inyección subcutánea) y el grupo de III sulfato de morfina con vitamina E (60 mg/kg/día por vía oral). Posteriormente, los animales superovularon con PSMG (10 unidades) y 10 unidades de HCG. El día siguiente, los animales fueron sacrificados, los ovocitos fueron aspirados desde cada tubo uterino. Los ovocitos recogidos fueron utilizados para determinar la apoptosis mediante el ensayo de TUNEL con el uso de microscopio de fluorescencia. El número de ovocitos recuperados fueron 121, 132, 86 y 114 en los grupos control y experimental I, II y III, respectivamente. El tratamiento con sulfato de morfina aumentó la apoptosis en los ovocitos un 17,44 por ciento, mientras que la apoptosis de los ovocitos fue 4,13 por ciento en los controles. La suplementación con vitamina E en los ratones tratados con sulfato de morfina redujo la apoptosis de los ovocitos en 7,01 por ciento. Este estudio demostró que la morfina puede aumentar la apoptosis en los ovocitos y el tratamiento vitamina E redujo significativamente la apoptosis en los ovocitos de ratones tratados con sulfato de morfina.

Correlation between hyaluronan binding assay and TUNEL assay for sperm DNA integrity identification.

Objective: To study the correlation between the percentages of HA-unbound sperm and DNA fragmented sperm by TUNEL assay. Methods: The semen residue from semen analysis was tested by HBA and TUNEL assay. Results: The mean age of patients included in the study was 34.8 years (± 3.7 years). The proportion of HA-unbound sperm ranged from 11.3% to 24.2%, with a mean of 17.08% (± 3.24%). The range of TUNEL positive in semen samples was 2% to 11.75%, with a mean of 5.78% (± 2.28%). Pearson’s correlation between two tests was 0.848 (p<0.01). Intraobserver variation of the results of HBA ranged from 3.3% to 7.6%, with a mean of 6.23% (± 1.11%). Intraobserver variation of the results of TUNEL assay ranged from 0% to 6.9%, with a mean of 1.54% (± 2.7%). Agreement measuring of each test was determined by using intraclass correlation. The intraclass correlation coefficient of HBA and TUNEL assay were 0.970 (P<0.001) and 0.997 (P<0.001) respectively. Conclusion: As several studies have found, the binding capacity of sperm to HA is correlated with several sperm parameters. In this study, the strong correlation between the percentages of HA-unbound sperm and TUNEL positive sperm implies, furthermore, that the HA-bound sperm percentage correlates with low levels of DNA fragmentation.

Hypothermia Effect on Apoptotic Neuronal Death in Traumatic Brain Injury Model

OBJECTIVE: Many researchers believe that the hypothermia shows neuroprotective effect on brain injury. To understand the molecular mechanism of the hypothermic treatment, this study investigated its effects on the expression of cell death or survival related proteins such as p53, Bcl-2 and Bax in the rat traumatic brain injury(TBI) model. METHODS: Twenty rats (Spraque Dawley, 200~250g) were subjected to the brain injury of moderate severity (2.4~2.6atm) using the fluid percussion injury device and five rats were received only same surgery as controls. During 30minutes after the brain injury, the hypothermia group was maintained the body temperature around 34 degrees C while the control group were maintained that of 36 degrees C. Five rats in each group were sacrificed 12h or 24h after brain injury and their brain sections was analyzed for physical damages by H-E stains and the extent of apoptosis by TUNEL assay and immunohistochemical stains. The tissue damage after TBI was mainly observed in the ipsilateral cortex and partly in the hippocampus. RESULTS: Apoptosis was observed by TUNEL assay and the Bax protein was detected in both sample which harvested 12h and 24h after TBI. In the hypothermia treatment group, tissue damage and apoptosis were reduced in HE stains and TUNEL assay. In hypothermia treatment group rat shows more expression of the Bcl-2 protein and shows less expression of the Bax protein, at both 12h and 24h after TBI. CONCLUSION: These results show that the hypothermia treatment is an effective treatment after TBI, by reducing the apoptotic process. Therefore, it could be suggested that hypothermia has a high therapeutic value for treating tissue damages after TBI.

Effects of Mitomycin C on Lens: in Vitro Study

PURPOSE: Cataract could occur as a complication of mitomycin C (MMC) used to increase the success rate of glaucoma surgery. Thus, the effects of MMC on cellular growth inhibition and apoptosis induction were examined by the culturing lens epithelial cells and the morphologic change in cells when MMC induced apoptosis. METHODS: Cellular morphology was observed after treating the mouse lens epithelial cell line, alpha-TN4, with MMC at different concentrations. Cellular toxicity was measured using LDH. Furthermore, IC50 inhibiting cellular proliferation and IC50 inducing apoptosis were measured. Staining and TUNEL assay were done to define apoptosis. The lens shape was observed under a transmission electron microscope, and electrophoresis was performed investigate the change in protein level when cataract was induced by MMC. RESULTS: The lens epithelial cells were atrophic even at low concentrations of MMC and more severe cellular changes along with reduced survival rate were seen at higher concentrations. According to the results of measuring cellular toxicity using LDH, the amount of LDH increased with increasing concentrations of MMC. The results of staining showed the findings of apoptosis of cells with orange-colored, compressed nuclei at low concentrations of MMC. Also on microscopic observation, the compression of chromosomes and fragmentation of nuclei were seen. Furthermore, the number of the high molecularr 53 KDa protein was increased among lens proteins of fertilized egg. CONCLUSIONS: These results demonstrated that MMC induced apoptosis of lens epithelial cells; the formation of cataract occurred in subcapsular area and the equator region; and denatuation of lens proteins and high molecular weight 53 KDa protein were increased.

Effect of radiation dosage changes on the cell viability and the apoptosis induction on normal and tumorigenic cells / 대한구강악안면방사선학회지

PURPOSE: The study was aimed to detect the differences in the cell viability and the apoptosis induction after irradiation on normal and tumorigenic cells. MATERIALS AND METHODS: The study, that was generated for two human normal cells(RHEK, HGF-1) and two human tumor cells(KB, HT-1080), was tested using MTT assay at 1 day and 3 day after irradiation and TUNEL assay under confocal laser scanning microscope at 1 day after irradiation. Single irradiation of 0.5, 1, 2, 4, and 8 Gy were applied to the cells. The two fractions of 1, 2, 4, and 8 Gy were separated with a 4 hour time interval. The irradiation was done with 5.38 Gy/min dose rate using Cs-137 irradiator at room temperature. RESULTS AND CONCLUSIONS: 1. In 3-day group, the cell viability of HGF-1 cell was significantly decreased at 2, 4 and 8 Gy irradiation, the cell viability of KB cell was significantly decreased at 8 Gy irradiation and the cell viability of HT-1080 cell was significantly decreased at 4 and 8 Gy irradiation. 2. There was significant difference between RHEK and KB cell line in the cell viability of 3-day group at 8 Gy irradiation. There was significant difference between RHEK and HGF-1 cell line in the cell viability of 3-day group at 4 and 8 Gy irradiation. 3. There was a significantly decreased cell viability in 3-day group than those in 1-day group at 2, 4 and 8 Gy on HGF-1 cell, at 4 and 8 Gy on HT-1080 cell, at 8 Gy on KB cell. 4. We could detect DNA fragmented cells only on KB cell. Number of apoptotic cells of KB cell was significantly increased at 4 and 8 Gy irradiation. However, there was no correlation between cell viability and apoptosis. 5. On all 4 cell lines, there were no differences between single and split irradiation method in cell viability and apoptosis.