Sex Differences of Neutrophil Extracellular Traps on Lipopolysaccharide-Stimulated Human Neutrophils.

Objective: Sex differences exist in sepsis, but the commitment of neutrophils to these differences remains unclear. Neutrophil extracellular traps (NETs) function to remove pathogens, yet excessive NETs release can contribute to organ damage. This study explores effects of the gender hormones on endotoxin-induced NETs using neutrophils from both male and female sources. Methods: Blood samples were collected from healthy volunteers. Isolated neutrophils were seeded in collagen-coated cell culture plates, and NETs were induced by lipopolysaccharide (LPS) treatment. After 15 minutes of LPS treatment, 17ß-estradiol (0.03-272.4 ng/mL), testosterone enanthate (0.01-10 ng/mL), dimethyl sulfoxide, or ethanol (vehicle control) was added to the plates. These were incubated for three hours at 37°C with 5% CO2. Neutrophil extracellular traps formation was assessed using immunofluorescence staining. Results: Lipopolysaccharide-induced NETs formation was significantly greater in females than in males. In male-derived neutrophils, 17ß-estradiol at above the blood concentrations significantly suppressed LPS-induced NETs. No effect was seen while using testosterone enanthate to NETs at any concentration. In female-derived neutrophils, 17ß-estradiol, which was near to the highest concentration of non-pregnant women's blood, tended to increase NETs. Testosterone enanthate, which was near to female blood concentration, significantly promoted NETs. Conclusions: Sex differences existed in LPS-induced NETs of human neutrophil. In males, high concentrations of 17ß-estradiol administration may have a suppressive effect on excessive NETs during infection. In females, endogenous gender hormones may promote NETs during infection. Sex differences in neutrophils may need to be considered in organ damage owing to NETs excess such as sepsis.

A hyaluronic acid-containing reagent compatible with glass-bottom dishes and capable of sustained binding of human spermatozoa.

OBJECTIVE: Commercial products currently available for sperm selection utilizing hyaluronic acid (HA) binding prior to intracytoplasmic sperm injection (ICSI) are widely used but have some disadvantages. To potentially circumvent these limitations, we compared ICSI using a self-made hyaluronic acid (smHA) reagent with ICSI using SpermSlow. METHODS: The binding of the reagents to spermatozoa on plastic- or glass-bottom dishes was quantitated using spermatozoa that were isolated by density-gradient centrifugation and swim-up procedures (N = 10/group). Additionally, we investigated the relationship between the HA reagent used prior to ICSI and clinical outcomes after assisted reproduction with HA-ICSI (N = 81). RESULTS: The smHA reagent exhibited extremely stable binding to human spermatozoa. The binding time of spermatozoa was significantly longer in the smHA reagent than in SpermSlow on both plastic and glass dishes (plastic: 60.0 ± 0.0 min vs. 2.7 ± 5.9 min, P < 0.001; glass: 60.0 ± 0.0 min vs. 2.5 ± 1.8 min, P < 0.001). There were no significant differences in the normal fertilization rate between HA-ICSI with the smHA reagent (128/160, 80.0%) and HA-ICSI with SpermSlow (171/231, 74.0%, P = 0.184). The frequency of the blastocyst development from the HA-ICSI-derived zygote was significantly higher with the smHA reagent (74/101, 73.3%) than with SpermSlow (76/131, 58.0%, P = 0.019). The rates of biochemical pregnancy, clinical pregnancy, fetal heart movement, live birth, and miscarriage were not significantly different between HA-ICSI with the smHA reagent and HA-ICSI with SpermSlow. CONCLUSIONS: The blastulation rate was higher for HA-ICSI with the smHA reagent as compared with SpermSlow. Clinical outcomes, excluding blastulation, after HA-ICSI were the same using smHA reagent and using SpermSlow. Spermatozoa binding to the smHA reagent was not attenuated over a 60-min time course. In conclusion, this reagent may shorten and simplify HA-ICSI procedures because smHA can be used with any dish material, making it easier to observe the spindle or assess intracytoplasmic morphology.

Equol, a soybean metabolite with estrogen-like functions, decreases lipopolysaccharide-induced human neutrophil extracellular traps in vitro.

OBJECTIVE: Neutrophil extracellular traps (NETs) defend against acute infections. However, their overexpression causes organ failure during sepsis. Control of NET formation may improve the outcomes of patients with sepsis. Equol, a soybean isoflavone, is a female hormone analog, which prevents inflammation. We evaluated the effects of equol on NET formation in human neutrophils during inflammatory stimulation in vitro. METHODS: Healthy volunteers provided blood samples. An enzyme-linked immunosorbent assay (ELISA) assessed serum equol concentrations. NET formation in neutrophils was induced by lipopolysaccharide (LPS) treatment. ELISA quantified DNA-binding elastase, and immunostaining assessed NET formation. Reverse-transcription quantitative PCR and western blotting detected G-protein-coupled receptor 30 (GPR30) or peptidyl arginine deiminase 4 (PAD4) expression. Flow cytometry assessed neutrophil phagocytic ability with inactivated Escherichia coli. RESULTS: In neutrophils derived from males with low-serum equol levels (low-serum equol group), equol significantly decreased DNA-binding elastase levels and NET formation. Equol did not decrease NETs in neutrophils from males with high-serum equol levels. GPR30 expression of neutrophils was higher in the low-serum than in the high-serum equol group. PAD4 mRNA levels and nuclear PAD4 protein expression also decreased than the vehicle control in the low-serum equol group. Equol did not alter the phagocytic ability of neutrophils. In neutrophils from young females, equol had no inhibitory effect on NET formation. CONCLUSIONS: Equol decreases LPS-induced NET formation in neutrophils from males via inhibition of PAD4 expression. Our findings provide a rationale for investigating a new therapeutic approach using equol to control neutrophil activity during sepsis.

Electrical stimulation facilitates NADPH production in pentose phosphate pathway and exerts an anti-inflammatory effect in macrophages.

Macrophages play an important role as effector cells in innate immune system. Meanwhile, macrophages activated in a pro-inflammatory direction alter intracellular metabolism and damage intact tissues by increasing reactive oxygen species (ROS). Electrical stimulation (ES), a predominant physical agent to control metabolism in cells and tissues, has been reported to exert anti-inflammatory effect on immune cells. However, the mechanism underlying the anti-inflammatory effects by ES is unknown. This study aimed to investigate the effect of ES on metabolism in glycolytic-tricarboxylic acid cycle (TCA) cycle and inflammatory responses in macrophages. ES was performed on bone marrow-derived macrophages and followed by a stimulation with LPS. The inflammatory cytokine expression levels were analyzed by real-time polymerase chain reaction and ELISA. ROS production was analyzed by CellRox Green Reagent and metabolites by capillary electrophoresis-mass spectrometry. As a result, ES significantly reduced proinflammatory cytokine expression levels and ROS generation compared to the LPS group and increased glucose-1-phosphate, a metabolite of glycogen. ES also increased intermediate metabolites of the pentose phosphate pathway (PPP); ribulose-5-phosphate, rebose-5 phosphate, and nicotinamide adenine dinucleotide phosphate, a key factor of cellular antioxidation systems, as well as α-Ketoglutarate, an anti-oxidative metabolite in the TCA cycle. Our findings imply that ES enhanced NADPH production with enhancement of PPP, and also decreased oxidative stress and inflammatory responses in macrophages.

The role of death receptor signaling pathways in mouse Sertoli cell avoidance of apoptosis during LPS- and IL-18-induced inflammatory conditions.

Lipopolysaccharide (LPS) triggers infectious acute inflammation, and interleukin (IL)-18 is an inflammasome-mediated cytokine. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are high. Additionally, high-dose recombinant IL-18 (rIL-18) induced Leydig cell apoptosis. The blood-testis barrier formed by Sertoli cells protects testicular germ cells from both exogenous and endogenous harmful substances. However, the impact of LPS and IL-18 on Sertoli cells remained unclear. We stimulated TM4 cells, a mouse Sertoli cell line, with LPS (200 or 1000 ng/mL) or rIL-18 (0.1-100 ng/mL) at levels that induced Leydig cell apoptosis in our previous study and assessed caspase 3 cleavage and the mRNA expression of inflammatory cytokines and markers of apoptotic pathways (Tnfr1, Fasl, Fas, Fadd) after stimulation. Il6 mRNA was increased by LPS stimulation. Tnfα mRNA was increased by 200 ng/mL LPS but not 1000 ng/mL LPS. Fas was increased, but Fasl was decreased, by LPS. LPS had little influence on Tnfr1 or Fadd mRNA expression and did not induce apoptosis. Il18 mRNA was not increased, and Il18r1 was significantly decreased following LPS treatment. Treatment with rIL-18 increased Il18r1 mRNA and induced inflammation, but decreased Tnfr1 and had little influence on apoptosis, as indicated by Tnfα, Fasl, Fas, Fadd and cleaved caspase 3. These results suggested that Sertoli cells do not easily undergo apoptosis despite strong inflammatory stimuli. Additionally, Sertoli cells may resist inflammation and play a larger role in protecting testicular homeostasis than other component cells of the testis.

The migration speed of nucleolar precursor bodies in pronuclei affects in vitro fertilization-derived human embryo ploidy status and live birth.

Purpose: To study the relationship between clinical outcomes after assisted reproduction and the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN). Methods: NPB migration speed, embryo ploidy status, and live birth (LB) were retrospectively analyzed in IVF-derived zygotes. The central coordinates of the mPN, fPN, and NPBs were noted at multiple timepoints. The migration distance of NPBs between two sequential images was measured to calculate NPB migration speed. Results: The NPB migration speeds in mPN and fPN were significantly faster in euploid zygotes than in aneuploid zygotes. In multivariate logistic analysis, NPB migration speed in mPN and the female age were associated with euploidy. The NPB migration speeds in mPN and fPN were also significantly faster in zygotes that led to LB than in zygotes that led to no pregnancy. In a receiver operating characteristic curve analysis of LB by NPB migration speed in mPN, the cut-off value was 3.74 µm/h (AUC: 0.825, 95%CI: 0.688-0.963). When the zygotes were categorized by this cut-off value, there were significantly more LBs in zygotes with migration speed ≥ the cut-off (78.9% vs. 21.1%). Conclusions: Zygotes with quickly migrating NPBs demonstrated the developmental potential to become a baby.

Monophasic Pulsed Current Stimulation of Duty Cycle 10% Promotes Differentiation of Human Dermal Fibroblasts into Myofibroblasts.

OBJECTIVE: Many clinical trials have shown the therapeutic effects of electrical stimulation (ES) in various conditions. Our previous studies showed that ES (200 µA and 2 Hz) promotes migration and proliferation of human dermal fibroblasts (HDFs). However, the effective duty cycle and the effect of ES on myofibroblast differentiation are unclear. This study aimed to investigate the relationship between duty cycle and myofibroblast differentiation. METHODS: HDFs were subjected to ES (200 µA and 2 Hz) for 24 h with the duty cycle adapted at 0% (control), 10%, 50%, or 90%. α-smooth muscle actin (SMA) and transforming growth factor (TGF)-ß1 mRNA and α-SMA protein expressions were assessed. Collagen gel contraction was observed for 48 h after ES initiation and the gel area was measured. Cell viability and pH of culture medium were analyzed for cytotoxicity of the ES. RESULTS: Cell viabilities were decreased in the 50% and the 90% groups but ES did not influence on pH of culture media. ES with a duty cycle of 10% significantly promoted the mRNA expression of α-SMA and TGF-ß1. α-SMA protein expression in the 10% group was also significantly higher than that of the control group. Collagen gel subjected to ES with a duty cycle of 10% was contracted. CONCLUSION: Duty cycle can influence on myofibroblast differentiation and ES with a duty cycle 10% is the effective for wound healing.

Outcome of in vitro fertilization cycles with automatic time-lapse instrumentation combined with the early embryo viability assessment score.

AIM: To evaluate whether embryo selection using the early embryo viability assessment (EEVA) score increases the ongoing pregnancy rate of in vitro fertilization and intracytoplasmic sperm injection patients. METHODS: One hundred eighty-one patients whose serum anti mullerian hormone (AMH) level was greater than 0.5 ng/µL were enrolled in the study. All patients received oocyte retrieval repeatedly from June 2017 to January 2019. Transferred embryos were selected using the EEVA score and Veeck's criteria. We investigated the blastocyst rate according to the EEVA score and Veeck's criteria and also evaluated the clinical outcome following embryo transfer of the blastocysts. RESULTS: Blastocyst development rate (48.7%) and high-quality blastocyst (42.4%) of Veeck 1 was statistically higher than others. The blastocyst rate (71.4%) and high-quality blastocyst rate (60.0%) for EEVA 1 was the highest, and a correlation between the EEVA score and the blastocyst rate was also identified in cases younger than 40 years. Blastocyst rate of EEVA 1 + 2 (69.8% 208/298) was statistically higher than that of Veeck 1 + 2 (40.1% 317/791) (p < 0.05) and high-quality blastocyst rate of EEVA 1 + 2 (50.0% 104/208) was also higher than that of Veeck 1 + 2 (36.6% 117/320) (p < 0.05). However, there was a significant correlation between EEVA and the pregnancy rate and pregnancy rate of EEVA 1 + 2 showed no statistical difference compared with Veeck 1 + 2. CONCLUSIONS: Although it remains to be answered whether a computer can substitute Veeck's classification, the EEVA score could be a viable alternative to predict the blastocyst rate and to select those high-potential embryos that improve the pregnancy rate.

Migration speed of nucleolus precursor bodies in human male pronuclei: a novel parameter for predicting live birth.

PURPOSE: To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction. METHODS: The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction. RESULTS: The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 µm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid. CONCLUSION: The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection.

Interleukin-18 levels and mouse Leydig cell apoptosis during lipopolysaccharide-induced acute inflammatory conditions.

Interleukin (IL)-18 is an inflammasome-mediated cytokine produced by germ cells, Leydig cells, and resident macrophages that is indispensable in the maintenance of homeostasis in the testis. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are very high. However, the impact of acute inflammation and IL-18 on Leydig cells remained unclear. TM3 cells, a mouse Leydig cell line, and RAW264.7 cells, a mouse macrophage cell line, were stimulated with lipopolysaccharide (LPS) or recombinant IL-18 (rIL-18). We assessed the expression of inflammatory cytokines, caspase cleavage, and markers of apoptotic pathways. In Leydig cells, caspase 3 cleavage was increased and death-receptor-mediated apoptotic pathways were activated after LPS stimulation. However, LPS stimulation did not increase IL-18 expression in the Leydig cell line. When high-dose rIL-18 was administered to the Leydig cell line to mimic levels seem after inflammation, rIL-18 upregulated Tnf-α mRNA, Fadd mRNA, and Fas protein, promoted cleavage of caspase-8 and caspase-3, and induced apoptosis. Low-dose rIL-18 did not stimulate apoptosis. To determine if the high level of IL-18 seen in the testes after inflammation was derived from immune cells, we examined IL-18 protein expression in a macrophage cell line, RAW264.7. In contrast to the TM3 cells, IL-18 was significantly increased in RAW264.7 cells after LPS stimulation. These results suggest that high-dose IL-18 derived from macrophages is harmful to Leydig cells. Reducing the overexpression of IL-18 could be a new therapeutic approach to prevent Leydig cell apoptosis as a result of acute inflammation.

Failure of complete hatching of ICSI-derived human blastocyst by cell herniation via small slit and insufficient expansion despite ongoing cell proliferation.

PURPOSE: To assess the effect of intracytoplasmic sperm injection (ICSI) on embryo hatching and visualise the effects of zona thinning (ZT) on the embryo using time-lapse monitoring. METHODS: In vitro fertilisation (IVF) (n = 178) and ICSI (n = 110)-derived cryopreserved blastocysts were donated by patients who previously had a baby. This study investigated the impacts of IVF, ICSI, laser-assisted hatching by ZT and formation of ICSI penetration trace on zona pellucida of IVF-derived blastocyst on blastcyst diameter, the estimated number of trophectoderm (TE) cells and completed hatching rate. RESULTS: The completed hatching rate and diameters of the completely hatched blastocysts at hatching commencement and at the maximum expansion were significantly greater in the IVF than in ICSI groups. The completed hatching rate significantly increased with ZT in both groups. The maximum diameters of the completely hatched blastocysts were significantly smaller in the ZT than in non-ZT groups. The estimated TE cell numbers increased from hatching commencement to their maximum expansion points. The incompletely hatched ICSI-derived blastocysts intermittently herniated cells via small slits until degeneration. The completed hatching rate significantly decreased by the formation of ICSI penetration trace on zona pellucida of IVF-derived blastocyst. CONCLUSION: ICSI-derived blastocysts intermittently release proliferating cells and extracted TE cells and/or inner cell masses via a small slit; thus, blastocyst expansion is not sufficiently increased, leading to a reduced complete hatching rate. Therefore, the ICSI penetration trace potentially has negative effects on blastocyst expansion process in vitro and is a risk factor for the failure of completed hatching.

Association of follicular fluid volume with membrane stretchability of human metaphase II oocytes following the gonadotropin-releasing hormone agonist protocol during intracytoplasmic sperm injection.

PURPOSE: The authors previously revealed the association of the follicular fluid (FF) volume with oolemma stretchability following the gonadotropin-releasing hormone (GnRH) antagonist protocol during intracytoplasmic sperm injection (ICSI). However, the impact of the GnRH agonist protocol on oolemma stretchability remains unclear. METHODS: Data that were obtained from 74 ICSI cycles were reviewed retrospectively. Controlled ovarian stimulation was performed in accordance with the short GnRH agonist protocol. Each follicle was individually aspirated and assigned to one of six groups, according to the FF volume. The oolemma stretchability during ICSI was evaluated by using a mechanical stimulus for oolemma penetration; that is, oolemma penetration with or without aspiration (high vs low stretchability, respectively). RESULTS: The incidence of low oolemma stretchability was significantly higher in the <1.0 mL group than that in the ≥1.0 mL group. The normal fertilization rate was significantly lower in the <1.0 mL group than that in the 2.0-<3.0 mL group. The rate of blastocyst development was lower in the <1.0 mL group than that in the 3.0-<4.0 mL group. CONCLUSION: The FF volume potentially was associated with metaphase II oolemma stretchability, fertilization, and blastocyst development.

The association of follicular fluid volume with human oolemma stretchability during intracytoplasmic sperm injection.

OBJECTIVE: Oocyte degeneration often occurs after intracytoplasmic sperm injection (ICSI), and the risk factor is low-quality oocytes. The follicular fluid (FF) provides a crucial microenvironment for oocyte development. We investigated the relationships between the FF volume aspirated from individual follicles and oocyte retrieval, oocyte maturity, oolemma stretchability, fertilization, and development. METHODS: This retrospective study included data obtained from 229 ICSI cycles. Ovarian stimulation was performed according to a gonadotropin-releasing hormone antagonist protocol. Each follicle was individually aspirated and divided into six groups according to FF volume (<1.0, 1.0 to <2.0, 2.0 to <3.0, 3.0 to <4.0, 4.0 to <5.0, and ≥5.0 mL). Oolemma stretchability during ICSI was evaluated using a mechanical stimulus for oolemma penetration, that is, the stretchability was assessed by oolemma penetration with aspiration (high stretchability) or without aspiration (low stretchability). RESULTS: Oocyte retrieval rates were significantly lower in the <1.0 mL group than in the ≥1.0 mL groups (46.0% [86/187] vs. 67.5%-74.3% [172/255 to 124/167], respectively; p<0.01). Low oolemma stretchability was significantly more common in the <1.0 mL group than in the ≥1.0 mL groups during ICSI (22.0% [13/59] vs. 5.8%-9.4% [6/104 to 13/139], respectively; p=0.018). There was a relationship between FF volume and oolemma stretchability. However, there were no significant differences in the rates of fertilization, cleavage, ≥7 cells at day 3, and blastocyst development among all groups. CONCLUSION: FF volume is potentially associated with the stretchability of metaphase II oolemma during ICSI. Regarding oolemma stretchability, ensuring a uniform follicular size during ovarian stimulation is crucial to obtain good-quality oocytes.

A lard-rich high-fat diet increases hepatic peroxisome proliferator-activated receptors in endotoxemic rats.

BACKGROUND: Diets high in saturated fatty acids activate chronic inflammation. We previously reported that, in even acute inflammation caused by lipopolysaccharide (LPS), liver injury was exacerbated in rats fed a lard-rich diet. Peroxisome proliferator-activated receptors (PPARs) are related to inflammation and are also key regulators of lipid metabolism. In this study, we examined effects of high-fat diet on liver injury and hepatic lipid metabolism during endotoxemia, measuring hepatic PPARs and other markers. MATERIALS AND METHODS: Male Wistar rats were fed a high-fat diet (HFD, 60 kcal% fat) or control diet (CD, 10 kcal% fat) for 4 or 12 wk, injected with LPS and sacrificed at 0, 1.5, or 6 h. Analyses included plasma aspartate transaminase (AST) and alanine transaminase (ALT) levels, messenger RNA (mRNA) and protein levels of hepatic PPARα and PPARγ, and mRNA levels of enzymes related to fatty acid oxidation and synthesis. RESULTS: Endotoxemic rats on HFD for 12 wk, but not 4 wk, had higher mRNA and protein levels for hepatic PPARs, than did those on CD (P < 0.01-0.05). Similarly, these rats had increased mRNA expression of hepatic fatty acid oxidation- and synthesis-related enzymes (P < 0.01-0.05). Rats injected with LPS had more severe liver injury, indicated by plasma AST/ALT, if on the HFD for 12 wk, compared with for 4 wk. CONCLUSIONS: Consumption of a lard-rich diet for 12 wk worsened liver injury and increased hepatic PPARα and PPARγ expression in endotoxemic rats.

Interleukin-18 Reduces Blood Glucose and Modulates Plasma Corticosterone in a Septic Mouse Model.

BACKGROUND: Dysregulation of glucose metabolism, including hyperglycemia with insulin resistance, is commonly observed in critically ill patients. Interleukin-18 (IL-18) improves the insulin resistance associated with obesity, but the relationship between IL-18 and glucose metabolism in sepsis was unclear. The purpose of this study was to investigate the influence of IL-18 on hyperglycemia during sepsis. METHODS: Sepsis was induced using cecal ligation and puncture (CLP) in wild-type (WT) mice, IL-18 knockout (KO) mice, and IL-18 KO mice pretreated with recombinant IL-18. Blood glucose and plasma insulin, glucagon, and corticosterone were measured. The mRNAs for gluconeogenic enzymes (g6pc, pck1) and activation of insulin signaling were also analyzed. RESULTS: In both WT and IL-18 KO mice, CLP operation led to hyperglycemia that lasted longer (18 h) than after sham operation (6 h). Blood glucose levels in IL-18 KO mice were significantly higher than in WT mice, without alteration of insulin or glucagon levels. In IL-18 KO mice, insulin signaling in the liver and skeletal muscle was decreased during hyperglycemia as compared with WT mice without suppression of hepatic glucose production enzymes. Pretreatment with recombinant IL-18 reduced blood glucose levels after CLP. Additionally, corticosterone levels were higher after CLP in the presence of either endogenous or exogenous IL-18. CONCLUSION: IL-18 may reduce blood glucose by modulating insulin signaling in the liver during sepsis-induced hyperglycemia. IL-18 is an important factor associated with alterations in blood glucose during sepsis.

Percentage of motile spermatozoa at 22 hours after swim-up procedure: An indicator for intracytoplasmic sperm injection?

OBJECTIVE: The decision to use in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or split insemination (IVF-ICSI) in the first cycle is based on the number of motile sperm. Hence, total fertilization failure (TFF) often occurs during IVF cycles, despite normozoospermia. To investigate whether the cumulative motile swim-up spermatozoa percentage at 22 hours post-insemination (MSPPI) is an indicator for ICSI, we analyzed TFF, fertilization, blastocyst development, chemical pregnancy, clinical pregnancy, and live birth rates. METHODS: This prospective study was performed using data obtained from 260 IVF cycles. At 22 hours after insemination, the remaining swim-up spermatozoa were observed and divided into six groups according to MSPPI (<10%, 10% to <30%, 30% to <50%, 50% to <70%, 70% to <90%, and 90% to 100%). RESULTS: Regardless of the ejaculated motile sperm concentration (0.6-280×10(6)/mL motile spermatozoa), the incidence of TFF significantly increased when MSPPI was <10%, and the fertilization rate significantly decreased when MSPPI was <30%. We found that cumulative MSPPI correlated with the cumulative fertilization rate (Spearman correlation, 0.508, p<0.001). Regarding embryo development, we observed no significant differences in the rates of blastocyst development, chemical pregnancy, clinical pregnancy, or live birth among all groups. CONCLUSION: Our findings suggest that MSPPI is a viable indicator for split IVF-ICSI and ICSI. Taken together, by employing the MSPPI test in advance before IVF, ICSI, or split IVF-ICSI cycles, unnecessary split IVF-ICSI and ICSI may be avoided.

Endogenous interleukin 18 regulates testicular germ cell apoptosis during endotoxemia.

Orchitis (testicular swelling) often occurs during systemic inflammatory conditions, such as sepsis. Interleukin 18 (IL18) is a proinflammatory cytokine and is an apoptotic mediator during endotoxemia, but the role of IL18 in response to inflammation in the testes was unclear. WT and IL18 knockout (KO) mice were injected lipopolysaccharide (LPS) to induce endotoxemia and examined 12 and 48  h after LPS administration to model the acute and recovery phases of endotoxemia. Caspase activation was assessed using immunohistochemistry. Protein and mRNA expression were examined by western blot and quantitative real-time RT-PCR respectively. During the acute phase of endotoxemia, apoptosis (as indicated by caspase-3 cleavage) was increased in WT mice but not in IL18 KO mice. The death receptor-mediated and mitochondrial-mediated apoptotic pathways were both activated in the WT mice but not in the KO mice. During the recovery phase of endotoxemia, apoptosis was observed in the IL18 KO mice but not in the WT mice. Activation of the death-receptor mediated apoptotic pathway could be seen in the IL18 KO mice but not the WT mice. These results suggested that endogenous IL18 induces germ cell apoptosis via death receptor mediated- and mitochondrial-mediated pathways during the acute phase of endotoxemia and suppresses germ cell apoptosis via death-receptor mediated pathways during recovery from endotoxemia. Taken together, IL18 could be a new therapeutic target to prevent orchitis during endotoxemia.

Endogenous interleukin 18 suppresses hyperglycemia and hyperinsulinemia during the acute phase of endotoxemia in mice.

BACKGROUND: Hyperglycemia associated with insulin resistance is common among critically ill patients. Interleukin (IL)-18 has been linked with hyperglycemia and insulin resistance in chronic disease, but the relation between IL-18 and insulin resistance during critical illness was unexplored. This study investigated whether IL-18 modulates hyperglycemia and insulin resistance during acute inflammation. METHODS: We injected lipopolysaccharide (LPS) 40 mg/kg into wild-type (WT) and IL-18 knockout (KO) mice to induce endotoxemia and examined insulin resistance and insulin-dependent signaling pathways during the acute phase. RESULTS: During the first hour after LPS treatment, IL-18 KO mice showed higher blood glucose and insulin and less insulin receptor substrate-1 and less phosphorylated Akt in the liver compared with WT mice. Interleukin-18 KO mice exhibited better survival after LPS treatment. CONCLUSIONS: The findings suggest that endogenous IL-18 may attenuate hyperglycemia and modulate insulin signaling in liver. Accordingly, IL-18 may modulate glucose tolerance during acute inflammation.

Acetic acid fermentation of acetobacter pasteurianus: relationship between acetic acid resistance and pellicle polysaccharide formation.

Acetobacter pasteurianus strains IFO3283, SKU1108, and MSU10 were grown under acetic acid fermentation conditions, and their growth behavior was examined together with their capacity for acetic acid resistance and pellicle formation. In the fermentation process, the cells became aggregated and covered by amorphous materials in the late-log and stationary phases, but dispersed again in the second growth phase (due to overoxidation). The morphological change in the cells was accompanied by changes in sugar contents, which might be related to pellicle polysaccharide formation. To determine the relationship between pellicle formation and acetic acid resistance, a pellicle-forming R strain and a non-forming S strain were isolated, and their fermentation ability and acetic acid diffusion activity were compared. The results suggest that pellicle formation is directly related to acetic acid resistance ability, and thus is important to acetic acid fermentation in these A. pasteurianus strains.

Successful pregnancy after intracytoplasmic sperm injection with testicular spermatozoa transported only under refrigeration.

PURPOSE: This case report describes two successful pregnancies after intracytoplasmic sperm injection (ICSI) with testicular spermatozoa that were transported under refrigeration. METHODS: Two first-time couples consulted our clinic concerned about their primary infertility. No sperm were present in the semen samples from either of the husbands and they were referred to the urology department (UD) of a neighbouring hospital. At the UD, seminiferous tubules were obtained by testicular sperm extraction. The tissue samples were put in a centrifuge tube with phosphate-buffered saline at 6°C and placed with refrigerant in a cushioned styrofoam box that was then transported to our clinic. Immediately upon arrival at our clinic, testicular spermatozoa were extracted. On the same day, ovum pickup was performed and mature oocytes were extracted that were then inseminated by conventional ICSI. Fertilized eggs were cultured for 2 days, and then cleaved embryos were cryopreserved. In one case after 4 months and in the other case after 2 months of cryopreservation, the frozen-thawed embryos were transferred. RESULT: Both patients became pregnant and normal, healthy babies were born. CONCLUSIONS: These results suggest that cases of obstructive azoospermia can be treated with ICSI by refrigerated transport of the seminiferous tubules, in cooperation with a UD, in a small single departmental obstetrics and gynecology clinic.