Identifying optimal puncture position by a real-time image analysis for Piezo-ICSI: a prospective randomized sibling oocyte study.

RESEARCH QUESTION: Would the use of the intracytoplasmic sperm injection (ICSI) position detector (IPD) make it possible to identify the optimal puncture position on oolemma during Piezo-ICSI and reduce oocyte degeneration and unintentional membrane rupture (UMR)? DESIGN: This sibling oocyte study included 917 inseminated oocytes from 113 infertile patients undergoing Piezo-ICSI. Oocytes were randomly divided into two groups: with or without IPD. The rates of UMR, degeneration, fertilization and embryonic development were compared between the two groups. As a secondary analysis, non-IPD oocytes were retrospectively assessed as appropriate or non-appropriate injection sites and analysed alongside prospective 'appropriate' injections. RESULTS: The rates of UMR (7.0% versus 12.9%, P = 0.004) and degeneration (2.4% versus 6.1%, P < 0.01 = 0.008) were significantly lower in the IPD group than in the non-IPD group. No significant differences, however, were observed in the rates of fertilization (two pronuclei, 83.8% versus 78.9%), blastocyst formation (48.5% versus 48.8%) or good-quality blastocysts (22.5% versus 20.5%). Additionally, no significant differences were observed in the rates of pregnancy (29.4% versus 35.1%) or live births (26.5% versus 29.7%) in a single embryo transfer setting with or without IPD. Comparing all 'appropriate' injections with 'non-appropriate' injections also showed a significantly decreased rate of UMR and degeneration (both P ≤ 0.001). CONCLUSIONS: The present study demonstrated that a real-time image analysis during Piezo-ICSI markedly reduced oocyte degeneration by avoiding areas associated with a high risk of UMR. Therefore, IPD may increase the number of embryos available for treatment.

Optimal puncture position for ICSI can be detected by image analysis using Local Binary Pattern.

RESEARCH QUESTION: One of the problems during the intracytoplasmic sperm injection (ICSI) procedure is unintentional membrane rupture (UMR), which often predisposes to subsequent oocyte degeneration. Can the ICSI Position Detector (IPD) be useful in identifying the optimal puncture location to prevent UMR during ICSI? DESIGN: A total of 709 mature oocytes were included. Conventional ICSI was carried out and images were recorded by IPD; these were analysed retrospectively. RESULTS: Inseminated oocytes were retrospectively grouped according to the IPD, irrespective of whether oolemma was punctured at an area in which UMR is likely (non-appropriate group) or unlikely (appropriate group). In the appropriate group, rates of UMR (5.3% versus 18.2%) and degeneration (2.5% versus 8.7%) were significantly lower than those of the non-appropriate group, whereas rate of fertilization (87.1% versus 69.7%) was significantly higher than those of the non-appropriate group, respectively (P < 0.001). These differences remained even after propensity score matching to adjust for potential differences in characteristics between appropriate and non-appropriate groups. CONCLUSIONS: This study demonstrated that the IPD is useful to identify the optimal puncture location to circumvent UMR during the ICSI procedure, resulting in reduced UMR and oocyte degeneration, thereby, generating more embryos available for transfer or cryopreservation.

Rupture Prediction for Microscopic Oocyte Images of Piezo Intracytoplasmic Sperm Injection by Principal Component Analysis.

Assisted reproductive technology (ART) has progressed rapidly, resulting in a great improvement in the clinical pregnancy ratio. When applying the protocol of piezo intracytoplasmic sperm injection (Piezo-ICSI), it is very important to puncture the zona pellucida and the oocyte cytoplasmic membrane without rupturing the oocyte cytoplasmic membrane. Previous studies have shown that the poor extensibility of the oocyte cytoplasmic membrane might be closely related to rupture. However, no consensus has been reached regarding how the quality of the oocyte for extensible ability or rupture possibility affects the surfaces of the oocyte on the microscopic frames. We conducted this study to provide evidence that artificial intelligence (AI) techniques are superior for predicting the tendency of oocyte rupture before puncturing on Piezo-ICSI. To inspect it, we provided a retrospective trial of 38 rupture oocytes and 55 nonruptured oocytes. This study marked the highest accuracy of 91.4% for predicting oocytes rupture using the support-vector machine method of machine learning. We conclude that AI technologies might serve an important role and provide a significant benefit to ART.

Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer.

(Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Here, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n = 20) or duodenal cancer/bile duct cancer (n = 16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. PCR products were subcloned into T-vectors, and the sequences were then analyzed. Immunohistochemical and serologic analyses for Enterococcus faecalis infection were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue-associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.

Fatty Acid-Mediated Stromal Reprogramming of Pancreatic Stellate Cells Induces Inflammation and Fibrosis That Fuels Pancreatic Cancer.

OBJECTIVES: Pancreatic ductal adenocarcinoma is one of the deadliest diseases worldwide. Fatty acids (FAs) have properties that affect both cancer cells and tumor environment. We assessed the effects of FAs on malignant characteristics in a pancreatic cancer and pancreatic stellate cell (PSC) coculture model. This study aimed to clarify the FA signature of PSC-derived inflammation and fibrosis in vitro and in a clinicopathological analysis. METHODS: The in vitro model involved coculture of the human pancreatic cancer cell lines PANC-1 and MIA PaCa-2 with human PSCs. Clinical histological samples were analyzed to characterize the surgical margins of samples from patients who received distal pancreatectomies. RESULTS: The pancreatic cancer cells took up lipids from the culture media. Saturated and unsaturated FAs were required to induce inflammatory responses in human PSCs, and the cocultures showed fibrotic changes. Clinical samples from pancreatic ductal adenocarcinoma patients had more fatty and fibrotic changes in the normal tissue in the surgical margins than samples from noncancer patients. CONCLUSIONS: Inflammation and fibrosis levels were increased in pancreatic cancer specimens, supporting the in vitro observations and suggesting that PSCs contribute to pancreatic carcinogenesis. Pancreatic stellate cells thus represent a potential therapeutic target for suppressing stromal changes in pancreatic cancer.

Use of Mac-2 binding protein as a biomarker for nonalcoholic fatty liver disease diagnosis.

In contrast to patients with viral hepatitis, patients with nonalcoholic fatty liver disease (NAFLD) can progress to hepatocellular carcinoma during the initial stages of liver fibrosis. Development and implementation of noninvasive methods for diagnosis and progression prediction are important for effective NAFLD surveillance. Mac-2 binding protein (Mac-2bp) is a useful nonalcoholic steatohepatitis (NASH) diagnosis biomarker and a powerful prediction biomarker for NAFLD fibrosis stage. Wisteria floribunda agglutinin (WFA)-positive Mac-2bp (WFA+-M2BP) is a novel serum fibrosis biomarker for chronic hepatitis C that has clinical validity. Mac-2bp and WFA+-M2BP are also clinical NAFLD biomarker candidates. We examined the efficacy of Mac-2bp and WFA+-M2BP for NAFLD assessment using patients with biopsy-proven NAFLD (n = 510; NAFLD cohort) and subjects who received a health check-up (n = 2,122; check-up cohort). In the NAFLD cohort, we set the fibrosis predicting cutoff values as 1.80 (F1), 2.21 (F2), and 2.24 µg/mL (F3). In the subjects with fatty liver from the check-up cohort (n = 1,291), the serum Mac-2bp levels were >1.80 µg/mL in 38.6% of the subjects (n = 498), and >2.24 µg/mL in 24.6% of the subjects (n = 318). The NAFLD cohort results indicated that Mac-2bp and WFA+-M2BP were equally useful for NASH diagnosis. During the early stages of fibrosis (F1, F2), the increase in Mac-2bp was statistically significant but WFA+-M2BP did not increase. Logistic regression analysis revealed that Mac-2bp was an independent determinant for the prediction of advanced fibrosis stage (≥F2), even when adjusted for WFA+-M2BP. Immunohistochemical staining of Mac-2bp revealed that hepatocytes strongly expressed Mac-2bp in patients with NAFLD. Conclusion: Our results indicated that hepatocyte-derived Mac-2bp would be a useful single biomarker for NASH diagnosis and fibrosis stage prediction in patients with NAFLD. (Hepatology Communications 2017;1:780-791).

Serum Mac-2 binding protein is a novel biomarker for chronic pancreatitis.

AIM: To determine the efficacy of Mac-2 binding protein (Mac-2bp) for diagnosis of chronic pancreatitis. METHODS: Fifty-nine healthy volunteers (HV), 162 patients with chronic pancreatitis (CP), and 94 patients with pancreatic ductal adenocarcinoma (PDAC) were enrolled in this study. We measured serum Mac-2bp using our developed enzyme-linked immunosorbent assay kit. Additional biochemical variables were measured using an automated analyzer (including aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, triglyceride, C-reactive protein, and amylase levels) or chemiluminescent enzyme immunoassay (carbohydrate antigen 19-9 and carcinoembryonic antigen). The ability of Mac-2bp to predict CP diagnosis accurately was assessed using receiver operating characteristic (ROC) analyses. RESULTS: Serum Mac-2bp levels were significantly increased in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Area under the ROC curve values of Mac-2bp for the discrimination of CP from HV and PDAC were 0.727 and 0.784, respectively. Multivariate analyses demonstrated that serum Mac-2bp levels were independent determinants for CP diagnosis from HV and PDAC patients. Immunohistological staining showed that Mac-2bp was expressed faintly in the pancreas tissues of both CP and PDAC patients. Serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, and triglyceride levels were significantly higher in patients with CP or PDAC. Serum Mac-2bp levels were highly correlated with protein levels of alanine aminotransferase, γ-glutamyltransferase, and C-reactive protein, but not amylase, suggesting that the damaged liver produces Mac-2bp. CONCLUSION: Measurement of serum Mac-2bp may be a novel and useful biomarker for CP diagnosis as well as liver fibrosis in the general population.

Specific increase in serum core-fucosylated haptoglobin in patients with chronic pancreatitis.

BACKGROUND/OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all malignancies, and its diagnosis in early stages is the most important prognostic factor. Chronic pancreatitis (CP), a common background of PDAC occurrence, is morphologically defined as progressive pancreatic fibrosis and inflammation accompanied by pancreatic exocrine cell atrophy. We recently found that inflammation and fibrosis are independent characteristic histological changes in noncancerous lesions in PDAC patients despite the absence of a past history of clinical CP. Subclinical CP is an important background for PDAC occurrence. Therefore, there is an urgent need to develop a noninvasive and reliable biomarker for CP diagnosis. METHODS: Fifty-nine healthy volunteers (HV), 159 patients with CP, and 83 patients with PDAC were enrolled in this study. We measured serum total fucosylated haptoglobin (Fuc-Hpt) and core-Fuc-Hpt levels using lectin-antibody enzyme-linked immunosorbent assay kits that we developed. In these kits, total Fuc-Hpt and core-Fuc-Hpt were measured using Aleuria aurantia lectin and Pholiota squarrosa lectin, respectively. RESULTS: Serum Fuc-Hpt levels were significantly increased in CP patients compared to HV (P < 0.0001) and were further increased in PDAC patients (P < 0.0001). Interestingly, serum core-Fuc-Hpt levels were significantly higher in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Multivariate analyses demonstrated that total serum core-Fuc-Hpt was an independent determinant for CP diagnosis, but Fuc-Hpt was not. CONCLUSIONS: A dramatic change in oligosaccharides was observed in serum haptoglobin between CP and PDAC. Serum core-Fuc-Hpt may be a novel and useful biomarker for CP diagnosis.

Influence of beta-blockers on the myocardial mRNA expressions of circadian clock- and metabolism-related genes.

Daily rhythms are regulated by a master clock-system in the suprachiasmatic nucleus and by a peripheral clock-system in each organ. Because norepinephrine is one of the timekeepers for the myocardial circadian clock that influences cardiac metabolism, it is speculated that a beta-blocker may affect the circadian clock and metabolism in heart tissue. In this study, thirty mg/kg/day of propranolol (a lipophilic beta-blocker) or atenolol (a hydrophilic beta-blocker) was given orally to Wistar rats for 4 weeks. The mRNA expressions of Bmal1 and E4BP4 in heart tissue were suppressed by the beta-blockers. However, the mRNA expressions of these clock genes in the suprachiasmatic nucleus were unchanged. Myocardial mRNA expressions of lactate dehydrogenase a and pyruvate dehydrogenase kinase 4 were also suppressed by the beta-blockers. In addition, ATP content in heart tissue was significantly elevated by the beta-blockers throughout 24 hours. The effects of propranolol and atenolol did not differ significantly. This study showed for the first time that a beta-blocker affects myocardial clock gene expression. Propranolol and atenolol increased ATP content in heart tissue throughout 24 hours. The influences of beta-blockers may be negligible on the SCN, and may be independent of lipid solubility on heart tissue. It is well known that these drugs exert a protective effect against myocardial ischemia, which may be mediated by an increase in the preservation of myocardial ATP.

Effects of telmisartan and valsartan on insulin sensitivity in obese diabetic mice.

Telmisartan and valsartan have angiotensin II receptor blocking activity. Because telmisartan has also an agonistic action for peroxisome proliferators-activator receptor (PPAR)-γ, it is speculated that an effect of telmisartan on insulin sensitivity is different from that of valsartan, which lacks of PPAR-γ agonistic activity. To address the issue, effects of telmisartan and valsartan on insulin sensitivity, adipocytokines and PPAR-γ target genes were evaluated in obese diabetic mice. KK-A(y) mice were treated with telmisartan (5mg/kg) and valsartan (15 mg/kg), once daily for 3 weeks. Insulin tolerance test was performed on day 14, and plasma adiponectin concentration and mRNA expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in adipose tissues were measured on day 21. Time-course of plasma glucose level after the injection of insulin in mice with telmisartan was not significantly different from that of animals with valsartan. In addition, PPAR-γ antagonist did not diminished the improvement of insulin sensitivity by telmisartan. Telmisartan and valsartan elevated plasma adiponectin concentration and suppressed the mRNA expressions of TNF-α and IL-6 in adipose tissues. These variables of the telmisartan- and valsartan-treated groups did not significantly differ. Influence of telmisartan on the PPAR-γ target genes (ap2 and fatty acid synthase) mRNA expressions was not detected in adipose tissues under the present condition. These data suggest that the effect of telmisartan on insulin sensitivity is similar to that of valsartan, and a role of PPAR-γ-mediated stimuli is small in the telmisartan-induced improvement of insulin sensitivity.

Reduced histone H3K9 acetylation of clock genes and abnormal glucose metabolism in ob/ob mice.

Recent chronobiological studies found significant correlation between lack of clock function and metabolic abnormalities. We previously showed that clock gene expressions were dampened in the peripheral tissues of obese and diabetic ob/ob mice. However, the molecular mechanism of the disturbance remained to be determined. In this study, we demonstrated for the first time that acetylation levels of histone H3 lysine 9 (H3K9) at the promoter regions of clock genes, such as Dbp, Per2, and Bmal1, in the adipose tissue of ob/ob mice were significantly reduced compared with those of its control C57BL/6J mice. Treatment with histone deacetylase (HDAC) inhibitors increased Dbp, but not Per2 or Bmal1, mRNA expression in adipose tissue, and it decreased blood glucose in these animals. In addition, 2-deoxyglucose uptake activity was significantly suppressed by silencing Dbp expression in cultured adipocytes. These results suggest that reduced H3K9 acetylation and subsequent decreased mRNA expression of the Dbp gene in adipose tissue are involved in the mechanism of development of abnormal glucose metabolism in ob/ob mice.

Tissue-dependent alterations of the clock gene expression rhythms in leptin-resistant Zucker diabetic fatty rats.

Recent studies have demonstrated that circadian clocks are impaired in liver and adipose tissue of both leptin-deficient ob/ob and leptin-resistant KK-A(y) mice. Because impairment of peripheral clocks precedes metabolic abnormalities in ob/ob mice, leptin signaling might be important for modulating peripheral clocks. To assess this hypothesis, the authors determined daily mRNA expression profiles of clock genes Clock, Arntl, Per1, Per2, Cry1, Dbp, and Nr1d1 in several tissues of leptin-receptor-deficient Zucker diabetic fatty (ZDF) rats. Transcript levels of some of these genes around the respective peak times decreased significantly in the liver, but not in the suprachiasmatic nucleus, mesenteric adipose tissue, and heart, compared to those in control rats. In contrast, mRNA levels of Per1 and Dbp around the peak time increased in the aorta of ZDF rats. However, expression rhythms of these clock genes in serum-stimulated cultured cells isolated from the aorta of ZDF rats were quite similar to those in serum-stimulated aortic cells of control rats. These results show that systemic leptin signaling defect influences peripheral clocks in a tissue-dependent manner, suggesting the possibility that leptin indirectly modulates the clocks in at least a subset of peripheral tissues.

Influence of dosing time on the efficacy and safety of finasteride in rats.

Finasteride (FIN), a widely used medication for the treatment of androgen-dependent diseases, blocks the conversion of testosterone to a more potent androgen, dihydrotestosterone (DHT). In this study, we investigated a dosing time-dependent effect and safety of FIN in rats. Androgen receptor (AR) mRNA and nuclear protein levels exhibited clear daily rhythms with the peak during the dark period in the prostate and during the light period in the liver. Repeated oral administration of FIN (5 or 100 mg/kg) at 3 h after lights on (HALO) for 2 weeks decreased serum DHT concentration throughout a 24-h period, whereas the dosing of the agent at 15 HALO decreased its level only transiently even in the higher dose group. FIN caused laboratory abnormalities in the 3 HALO group but not in the 15 HALO group. However, the effect of FIN on the prostate weight was not influenced by the dosing time. These results suggest that the safety, but not effect, of FIN depends on its dosing time in rats. The dosing of FIN in the active period might be a rational dosage regimen, which is needed to be confirmed in human subjects.

Induction of ß-catenin by the suppression of signal regulatory protein α1 in K562 cells.

The signal regulatory protein (SIRP) α1 is a cell surface receptor expressed predominantly in monocytes, granulocytes, dendritic cells, as well as hematopoietic stem cells. In contrast, SIRPα1 expression is significantly reduced in the majority of myeloid malignancies. SIRPα1 is a negative regulator of signaling and its reduced expression is considered to play a role in the pathogenesis of these diseases through aberrant signaling. To identify SIRPα1 downstream target genes, we established SIRP α1-knockdown chronic myeloid leukemia K562 (K562SIRPα1KD) cells expressing reduced levels of SIRPα1 by stably transfecting SIRPα1 siRNAs. Microarray analysis demonstrated that several genes, including ß-catenin, were significantly induced in K562SIRPα1KD cells. Real-time PCR and Western blot analyses, confirmed the induction of this gene. Phosphorylation of Ser9 of glycogen synthesis kinase (GSK) -3ß, results in the inactivation of GSK-3ß, leading to the induction of ß-catenin. We found significant phosphorylation of extracellular signal-regulated kinase (ERK), Akt, as well as of GSK-3ß-Ser9, which may play a role in the up-regulation of ß-catenin in K562SIRPα1KD cells. To our knowledge, this is a first report demonstrating the relationships between SIRPα1 and ß-catenin in leukemia cells.

Impairment of peripheral circadian clocks precedes metabolic abnormalities in ob/ob mice.

Recent studies have demonstrated relationships between the dysfunction of circadian clocks and the development of metabolic abnormalities, but the chicken-and-egg question remains unresolved. To address this issue, we investigated the cause-effect relationship in obese, diabetic ob/ob mice. Compared with control C57BL/6J mice, the daily mRNA expression profiles of the clock and clock-controlled genes Clock, Bmal1, Cry1, Per1, Per2, and Dbp were substantially dampened in the liver and adipose tissue, but not the hypothalamic suprachiasmatic nucleus, of 10-wk-old ob/ob mice. Four-week feeding of a low-calorie diet and administration of leptin over a 7-d period attenuated, to a significant and comparable extent, the observed metabolic abnormalities (obesity, hyperglycemia, hyperinsulinemia, and hypercholesterolemia) in the ob/ob mice. However, only leptin treatment improved the impaired peripheral clocks. In addition, clock function, assessed by measuring levels of Per1, Per2, and Dbp mRNA at around peak times, was also reduced in the peripheral tissues of 3-wk-old ob/ob mice without any overt metabolic abnormalities. Collectively these results indicate that the impairment of peripheral clocks in ob/ob mice does not result from metabolic abnormalities but may instead be at least partially caused by leptin deficiency itself. Further studies are needed to clarify how leptin deficiency affects peripheral clocks.

Protective effect of amlodipine against osteoporosis in stroke-prone spontaneously hypertensive rats.

Hypertensive patients have an increasing risk of osteoporosis. A recent case-controlled study has demonstrated that anti-hypertensive therapy reduced a risk of fracture in these patients. In this study, we investigated whether amlodipine protects against the reduction in bone density in stroke-prone spontaneously hypertensive rats (SHR-sp). Oral dosing of amlodipine (0.5 and 3.0mg/kg/day) was started when SHR-sp were 3 months old, and continued for 3 months. At the end of the experiment, bone density of femur and serum concentrations of calcium, parathyroid hormone (PTH) and C-telopeptide of type I collagen (CTx), reflecting osteoclast activity, were measured. The bone density dose-dependently increased by the treatment with amlodipine. In addition, amlodipine reduced serum concentrations of calcium, PTH and CTx. This study showed that amlodipine prevents the reduction in bone density during the repeated dosing in SHR-sp. Amlodipine might exert its effect through a direct inhibition of osteoclast function and/or suppression of PTH secretion and subsequent inhibition of osteoclast activity.

Dosing-time dependent effect of dexamethasone on bone density in rats.

AIMS: While glucocorticoids are widely used to treat patients with various diseases, they often cause adverse effects such as bone fractures. In this study, we investigated whether the decrease in bone density induced by glucocorticoid therapy was ameliorated by optimizing a dosing-time. MAIN METHODS: Rats were administered with dexamethasone (Dex) orally (1mg/kg/day) for 6weeks at a resting or an active period. After the end of the treatment, bone density of femur, biomarkers of bone formation and resorption, and other biomedical variables were measured. KEY FINDINGS: Bone density of femur was significantly decreased by the 6-week treatment with Dex, and the degree of decrease in the 14 HALO (hours after light on) dosing group (an active period) was larger than that in the 2 HALO dosing group (a resting period). Although urinary calcium excretion was accelerated by Dex treatment, secondary hyperparathyroidism was not detected. Histomorphometry analysis showed that Dex suppressed bone resorption, which was larger in the 2 HALO than in the 14 HALO groups. These data indicate that Dex equally suppressed bone formation in the 2 and 14 HALO groups, but inhibited bone resorption more in the 2 HALO than in the 14 HALO groups. SIGNIFICANCE: This study shows that the decrease in bone density induced by Dex was changed by its dosing-time.

Cranberry juice suppressed the diclofenac metabolism by human liver microsomes, but not in healthy human subjects.

AIM: To investigate a potential interaction between cranberry juice and diclofenac, a substrate of CYP2C9. METHODS: The inhibitory effect of cranberry juice on diclofenac metabolism was determined using human liver microsome assay. Subsequently, we performed a clinical trial in healthy human subjects to determine whether the repeated consumption of cranberry juice changed the diclofenac pharmacokinetics. RESULTS: Cranberry juice significantly suppressed diclofenac metabolism by human liver microsomes. On the other hand, repeated consumption of cranberry juice did not influence the diclofenac pharmacokinetics in human subjects. CONCLUSIONS: Cranberry juice inhibited diclofenac metabolism by human liver microsomes, but not in human subjects. Based on the present and previous findings, we think that although cranberry juice inhibits CYP2C9 activity in vitro, it does not change the pharmacokinetics of medications metabolized by CYP2C9 in clinical situations.