Transwell Culture with Adipose Tissue-Derived Stem Cells and Fertilized Eggs Mimics the In Vivo Development of Fertilized Eggs to Blastocysts in the Fallopian Tube: An Animal Study.

Many countries, including Japan, are experiencing declining birth rates. Assisted reproductive technologies have consistently demonstrated good results in resolving infertility. Although the development of fertilized eggs into blastocysts has been recognized as a crucial step in assisted reproductive technologies, the involved mechanisms are currently unclear. Here, we established a new culture system for the in vitro development of fertilized eggs into blastocysts. In the Transwell culture system, the rate of blastocysts hatching from fertilized eggs cultured with adipose-derived stem cells (ASCs) was significantly higher than that of blastocysts cultured only with fertilized eggs. Gene ontology analysis revealed that the developed blastocysts displayed essential gene expression patterns in mature blastocysts. Additionally, when cultured with 3rd-passage ASCs, the developed blastocysts expressed the core genes for blastocyst maturation and antioxidant properties compared to those cultured only with fertilized eggs or cultured with 20th-passage ASCs. These results suggest that the Transwell culture system may imitate the in vivo tubal culture state for fertilized eggs. Exosomes derived from stem cells with stemness potential play a powerful role in the development of blastocysts from fertilized eggs. Additionally, the exosomes expressed specific microRNAs; therefore, the Transwell culture system resulted in a higher rate of pregnancy. In future, the extraction of their own extracellular vesicles from the culture medium might contribute to the development of novel assisted reproductive technologies.

Safety and Potential Effect of Intrauterine Infusion of Autologous Adipose Tissue-Derived Regenerative Cells in Patients With Implantation Failure: A Pilot Study.

BACKGROUND: Implantation failure due to thin endometrium has emerged as a major cause of infertility. In this study, we aimed to assess the safety and preliminary efficacy of adipose tissue-derived regenerative cells (ADRCs), a source of adipose-derived stem cells, in infertility patients with implantation failure. METHODS: Five infertile women with implantation failure despite artificial reproductive technology were enrolled in this study and treated with ADRCs via the intrauterine route. The primary outcome was the incidence of adverse events. Additional outcomes were endometrial thickness after ADRC treatment and pregnancy success after embryo transfer. RESULTS: There were no adverse events in any patient. There was no elevation of white blood cell count, C-reactive protein, or D-dimer levels. There was a significant difference in endometrial thickness in the secretory phase before versus after intrauterine transplantation of ADRCs (3.8 ± 1.3 mm versus 8.8 ± 2.8 mm, respectively; p<0.05). A gestational sac and fetal heartbeat were detected on transvaginal ultrasound in two of five patients. CONCLUSION: Intrauterine infusion of autologous ADRCs is a simple and safe procedure that may ameliorate the endometrial microenvironment in infertile women with implantation failure.

Clinical Prediction of Retained Products of Conception: Combining Obstetric History and Ultrasound for Improved Accuracy in Severe Postpartum Hemorrhage.

Background The current challenge is how to improve the management of postpartum hemorrhage (PPH) to reduce the maternal mortality rate further. This study aimed to investigate whether a combined specific obstetric history and ultrasonographic findings can improve the predictive accuracy of retained products of conception (RPOC) with severe PPH. Methods This retrospective study included 56 patients who were diagnosed with RPOC. We extracted the following clinical data: obstetric history of second-trimester miscarriage, the time at which there was clinical suspicion of RPOC after the previous pregnancy (TIME), grayscale ultrasonographic finding (RPOC long-axis length [SIZE]), and color Doppler ultrasonographic finding based on the Gutenberg classification (RPOC hypervascularity). In this study, we defined cases requiring blood transfusion therapy or transcatheter arterial embolization as severe PPH. The patients were divided into two groups according to the presence or absence of severe PPH. The predictors of severe PPH were evaluated using logistic regression models. Model A comprised a combination of second-trimester miscarriage and TIME, Model B comprised a combination of Model A and long-axis SIZE, and Model C comprised a combination of Model B and RPOC hypervascularity. Results The multivariable analysis showed that long-axis SIZE was the only significant predictor of severe PPH (odds ratio [OR], 10.38; 95% confidence interval [CI], 2.06-63.86) independent of second-trimester miscarriage, TIME, and RPOC hypervascularity. The c-statistic was higher in Model C (OR, 0.863; 95% CI, 0.731-0.936) than in Model A (OR, 0.723; 95% CI, 0.551-0.847) and Model B (OR, 0.834; 95% CI, 0.677-0.923). Conclusion Combining a specific obstetric history (second-trimester miscarriage and TIME) and ultrasonographic findings (long-axis SIZE and RPOC hypervascularity) improves the predictive accuracy of RPOC with severe PPH. This prediction model may be a useful clinical screening tool for RPOC with severe PPH.

Physical Properties of Ultrafine Bubbles Generated Using a Generator System.

BACKGROUND/AIM: Ultrafine bubbles (UFBs) have been extensively researched owing to their promising physical and biological properties. However, determining the lifespan or ideal concentration of UFBs for various biological events is challenging. This study aimed to determine the maximum concentration and longest lifespan of UFBs and to verify the validity of UFBs for assessing cell properties. MATERIALS AND METHODS: A generator system (HMB-H0150+P001, TOSSLEC Corporation Limited, Kyoto, Japan) generated UFBs using various gases. The size and concentration of UFBs in ultrapure water and cell culture medium were measured through a nanoparticle tracking analysis method. RESULTS: The UFB concentration increased when the generator operated in a time dependent manner. The mean size of UFBs was approximately 120 nm. In the UFB lifespan, the concentration decreased by approximately 30% within the first two weeks of generation and was stable for up to 6 months. The UFB size increased by approximately 20% within the first two weeks of generation and demonstrated minor changes until the 6th month. The number of cells differed significantly with various concentrations of nitrogen gas UFBs. CONCLUSION: The generator system can generate UFBs with multiple concentrations within a suitable temperature. Consequently, the solution containing UFBs could be widely acceptable in cell culture systems.

Cumulative Summation Analysis of Learning Curve for Robotic-assisted Hysterectomy in Patients With Gynecologic Tumors.

BACKGROUND/AIM: This study aimed to evaluate the learning curve and perioperative outcomes of robot-assisted hysterectomy (RAH). PATIENTS AND METHODS: We retrospectively analyzed data from 45 patients who underwent RAH using the da Vinci Xi surgical system. The learning curve was evaluated using the cumulative summation method. Demographic data and various perioperative parameters, including total operative time, docking time, and console time, were obtained from the medical records. RESULTS: Cumulative summation analysis indicated that proficiency regarding hysterectomy time was reached after 33 cases. There were two unique phases of the learning curve for console time: the introduction phase identified by the bottom point in the curve, and the proficient phase, identified by an upward line after the bottom point in the curve. There were no significant differences between the two phases in terms of patient age and body mass index. Total operative time, docking time, and console time were significantly decreased in the proficient phase compared with those in the introduction phase. There was a significant reduction in blood loss during operation in the proficient phase. The perioperative complication rates were 12.1% in the introduction phase and 0% in the proficient phase (p=0.5606). No blood transfusion or conversion to laparotomy was required in either phase. CONCLUSION: The introduction and proficient phases identified by cumulative summation analysis demonstrated progressive improvement of surgical performance in surgeons carrying out RAH.

Trophic and immunomodulatory effects of adipose tissue derived stem cells in a preclinical murine model of endometriosis.

Endometriosis, which exhibits enigmatic pathological features such as stromal fibrosis and proliferation of ectopic epithelial cells, is known as a refractory disease. Mesenchymal stem cells modulate the fibrosis in stromal tissues through their trophic and immunomodulatory properties. To investigate the potential of stem cells in treating endometriosis, we examined the secondary morphology and molecular alterations in endometriosis-like lesions after the administration of adipose tissue-derived stem cells (ASCs) to an experimental murine model of endometriosis. The infused ASCs were found integrated in the endometriosis-like lesions. Accompanied by the suppression of stromal fibrosis and proliferation of endometriotic epithelial cells, the infusion of ASCs with stemness potential (early passage of ASCs) suppressed the growth of endometriosis-like lesions and inhibited the expression of pro-inflammatory and pro-fibrotic cytokines, whereas no significant attenuation of endometriosis-like lesions occurred after the infusion of ASCs without stemness potential (late passage of ASCs). Accordingly, the trophic and immunomodulatory properties of ASCs may regulate fibrosis in endometriosis-like lesions, suggesting that regenerative medicine could be recognized as an innovative treatment for patients with endometriosis through the accumulation of evidence of preclinical efficacy.

Vaginal microbiome as a tool for prediction of chorioamnionitis in preterm labor: a pilot study.

Intra-amniotic infection (IAI) is a major cause of preterm birth with a poor perinatal prognosis. We aimed to determine whether analyzing vaginal microbiota can evaluate the risk of chorioamnionitis (CAM) in preterm labor cases. Vaginal discharge samples were collected from 83 pregnant women admitted for preterm labor. Based on Blanc's classification, the participants were divided into CAM (stage ≥ II; n = 46) and non-CAM (stage ≤ I; n = 37) groups. The 16S rDNA amplicons (V1-V2) from vaginal samples were sequenced and analyzed. Using a random forest algorithm, the bacterial species associated with CAM were identified, and a predictive CAM (PCAM) scoring method was developed. The α diversity was significantly higher in the CAM than in the non-CAM group (P < 0.001). The area under the curve was 0.849 (95% confidence interval 0.765-0.934) using the PCAM score. Among patients at < 35 weeks of gestation, the PCAM group (n = 22) had a significantly shorter extended gestational period than the non-PCAM group (n = 25; P = 0.022). Multivariate analysis revealed a significant difference in the frequency of developmental disorders in 3-year-old infants (PCAM, 28%, non-PCAM, 4%; P = 0.022). Analyzing vaginal microbiota can evaluate the risk of IAI. Future studies should establish appropriate interventions for IAI high-risk patients to improve perinatal prognosis.

MicroRNAs miR-4535 and miR-1915-5p in amniotic fluid as predictive biomarkers for chorioamnionitis.

BACKGROUND: This study was performed to investigate the clinical significance of miR-4535 and miR-1915-5p in severe chorioamnionitis. MATERIALS & METHODS: Amniotic fluid samples from 37 patients with severe chorioamnionitis were subjected to miRNA array analysis and ddPCR™. Diagnostic values were assessed using the receiver operating characteristic curve. The patients were separated into three groups according to Blanc's criteria. RESULTS: The expression of miR-4535 and miR-1915-5p was significantly correlated with the copy number of 16S rDNA, had extremely high diagnostic accuracy for severe chorioamnionitis, and was linked to maternal and fetal inflammation. CONCLUSION: miR-4535 and miR-1915-5p serve as promising biomarkers for the diagnosis of severe chorioamnionitis.

Adipose tissue-derived regenerative cells improve implantation of fertilized eggs in thin endometrium.

Aim: Embryo implantation and subsequent pregnancy depends on endometrial thickness. To investigate potential fertility strategies for women with thin endometrium, we explored the efficacy of adipose tissue-derived regenerative cells (ADRCs) on thin endometrium and embryo implantation in a mouse model. Materials & methods: ADRCs isolated from mouse subcutaneous fat were characterized by flow cytometry. Endometrium thickness, endometrial fibrosis, embryo implantation and angiogenesis factors were evaluated in uterine cavities of ethanol-induced thin endometrium mice with ADRC transplantation. Results: ADRCs included adipose-derived stem cells and some blood vessel component cells. ADRCs improved endometrial thickness, endometrial fibrosis and embryo implantation and augmented vascular endothelial growth factor expression in the mouse uterine. Conclusion: ADRCs may be a useful therapeutic strategy to improve fertility of women with thin endometrium.

Clinical Features of Recurrence in Patients Without Residual Tumour in Endometrial Cancer.

BACKGROUND/AIM: Initial treatment of endometrial cancer with surgery and platinum and taxane-based chemotherapy is often successful, but it remains unclear as to whether certain types of the disease relapse. The aim of this study was to identify the clinical features of recurrence in patients without residual tumour in endometrial cancer. PATIENTS AND METHODS: Clinical features, histological type, and time to recurrence were analyzed in 640 endometrial cancer patients without residual tumours. RESULTS: Of 640 patients, 517 were type I and 123 were type II. For type I, early recurrent (ER) disease and late recurrent (LR) disease were noted in 80 and 8 patients, respectively, and 97.5% of ER occurred within 2 years. After recurrence, 76.2% of ER and 50% of LR patients died. In type II, ER and LR were noted in 41 and 1 patients, respectively, and 97.6% of ER occurred within 2 years, of which 75.6% died after recurrence. One LR case died of disease. CONCLUSION: Most patients recurred within 2 years irrespective of clinical stage or type.

A partial supernumerary umbilical vein: a case report.

BACKGROUND: Abnormalities in the number of vessels can be found for both the umbilical artery and vein. We sometimes encounter cases of a decreased number of umbilical cord vessels, such as a single umbilical artery. In contrast, there may be an increase from three to four vessels within the umbilical cord. A supernumerary umbilical vein is particularly very rare, and it is generally found in combination with congenital anomalies. We report a case of a partial supernumerary umbilical vein. CASE PRESENTATION: The previous pregnancy of a 37-year-old healthy Japanese woman (gravida 2, para 1) had been uncomplicated, and the resulting child was alive and well. Prenatal examination at 36 weeks of gestation revealed the coexistence of a four-vessel part and a normal three-vessel part of the umbilical cord. A healthy female neonate weighing 2726 g was born at 38 weeks of gestation. The umbilical cord measured 40 cm in length; the four-vessel part continued to a distance of 18 cm from the surface of the infant's body, and the remaining umbilical cord comprised three vessels. On histological examination, the fetal side of the umbilical cord had two arteries and two veins, and the placental side had two arteries and one vein. Isolated supernumerary umbilical veins tend to be overlooked. We consider that it is important to evaluate the number of umbilical cord vessels in the second trimester using ultrasound combined with color Doppler in at least three sites: the insertion sites on both the fetal abdomen and placenta, and the free loop of the umbilical cord. CONCLUSIONS: Prenatal diagnosis of isolated supernumerary umbilical cord vessels tends to be overlooked. However, supernumerary vessels of the umbilical can be associated with fetal congenital anomalies. The number of vessels within the umbilical cord must be examined because the detection of such abnormalities may lead to the prenatal diagnosis of other congenital anomalies.

The combination of maternal blood and amniotic fluid biomarkers improves the predictive accuracy of histologic chorioamnionitis.

INTRODUCTION: This study was performed to determine whether the combination of maternal blood and amniotic fluid biomarkers can improve the predictive accuracy of histologic chorioamnionitis (HC). METHODS: This retrospective study included 80 singleton pregnant women who were suspected to have intrauterine infection and underwent measurement of two maternal blood biomarkers [maternal white blood cell count (mWBC) and maternal C-reactive protein level (mCRP)] and three amniotic fluid biomarkers [amniotic white blood cell count (aCell), amniotic glucose level (aGlucose), and amniotic lactate dehydrogenase level (aLDH)]. We divided the patients into two groups based on the presence or absence of HC and assessed the predictors of HC using logistic regression models: Model 1, combination of mWBC and mCRP; Model 2, combination of Model 1 and aGlucose; and Model 3, combination of Model 2, aCell, and aLDH. RESULTS: The multivariable analysis showed that aCell was the only significant predictor of HC [odds ratio, 1.24; 95% confidence interval (CI), 1.06-1.68] independent of mWBC, mCRP, aGlucose, and aLDH. The c-statistics were higher in Model 3 (0.803; 95% CI, 0.701-0.905) than Model 1 (0.634; 95% CI, 0.511-0.758) and Model 2 (0.785; 95% CI, 0.684-0.887). DISCUSSION: We found that the combination of maternal blood and amniotic fluid biomarkers can improve the predictive accuracy of HC. Therefore, our data provide relevant information to support counseling with regard to improving the predictive accuracy of HC in patients with suspected intrauterine infection.

Association of Serum HB-EGF Value and Response to Chemotherapy in Patients with Recurrent Ovarian Cancer.

BACKGROUND/AIM: Many anticancer agents including molecularly-targeted drugs have been developed for ovarian cancer. However, the prognosis of recurrent ovarian cancer remains extremely poor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is reported as a rational target for ovarian cancer therapy. Moreover, serum HB-EGF expression is recognized as a biomarker in patients with primary ovarian cancer. MATERIALS AND METHODS: We analysed serum samples with recurrent ovarian cancer at the Fukuoka University Hospital from April 2009 to March 2014. To assess the clinical significance of serum HB-EGF in recurrent ovarian cancer, the association between serum HB-EGF levels and prognosis in patients with recurrent ovarian cancer was examined using ELISA. RESULTS: Patients with high serum HB-EGF expression showed a significantly poor response to second-line chemotherapeutic agents compared with patients with low HB-EGF levels. CONCLUSION: HB-EGF expression in serum may be a potential therapeutic indicator for novel HB-EGF-targeted therapy in recurrent ovarian cancer.

Microbiome profile of the amniotic fluid as a predictive biomarker of perinatal outcome.

Chorioamnionitis (CAM), an inflammation of the foetal membranes due to infection, is associated with preterm birth and poor perinatal prognosis. The present study aimed to determine whether CAM can be diagnosed prior to delivery based on the bacterial composition of the amniotic fluid (AF). AF samples from 79 patients were classified according to placental inflammation: Stage III (n = 32), CAM; Stage II (n = 27), chorionitis; Stage 0-I (n = 20), sub-chorionitis or no neutrophil infiltration; and normal AF in early pregnancy (n = 18). Absolute quantification and sequencing of 16S rDNA showed that in Stage III, the 16S rDNA copy number was significantly higher and the α-diversity index lower than those in the other groups. In principal coordinate analysis, Stage III formed a separate cluster from Stage 0-I, normal AF, and blank. Forty samples were classified as positive for microbiomic CAM (miCAM) defined by the presence of 11 bacterial species that were found to be significantly associated with CAM and some parameters of perinatal prognosis. The diagnostic accuracy for CAM according to miCAM was: sensitivity, approximately 94%, and specificity, 79-87%. Our findings indicate the possibility of predicting CAM prior to delivery based on the AF microbiome profile.

HB-EGF Is a Promising Therapeutic Target for Lung Cancer with Secondary Mutation of EGFRT790M.

Advanced lung cancer is one of the most lethal malignancies. Many anticancer agents have been developed for lung cancer with epidermal growth factor receptor (EGFR) mutations, but its prognosis remains extremely poor. The development of molecularly-targeted therapies is required for patients with lung cancer with secondary mutation of the EGFR gene. In this study, in order to assess the validity of heparin-binding EGF-like growth factor (HB-EGF) as a therapeutic target for lung cancer with EGFR mutation, we examined the antitumor effects of a specific inhibitor (cross-reacting material 197; CRM197) on lung cancer cells with EGFR mutation. HB-EGF was the most predominantly expressed EGFR ligand in lung cancer cells with EGFR mutation. CRM197 induced significant cell apoptosis and marked suppression of tumorigenicity in lung cancer cells with single or double mutation of EGFR. These results suggest that HB-EGF is a rational target for the treatment of lung cancer with EGFR mutation.

Antitumour Effects of Intravenous Administration of BK-UM, a Novel Inhibitor of HB-EGF, in Ovarian Cancer Therapy.

BACKGROUND: Patients with ovarian cancer with high levels of heparin-binding epidermal growth factor-like growth factor have a poor prognosis. Here we assessed the pharmacokinetics and tumour-inhibiting effects of cross-reacting material 197, produced commercially as BK-UM, and examined the efficacy and safety of its intravenous (i.v.) administration. MATERIALS AND METHODS: BK-UM was administered to rats, and its serum levels were measured. Ovarian cancer cell lines were either intraperitoneally (i.p.) or subcutaneously administered into mice, to establish a mouse model of ovarian cancer. BK-UM was then administered i.p. or i.v., and its tumour-inhibiting effects were examined. RESULTS: Higher maximum serum concentration (Cmax) values resulted from i.v. administration, whereas longer time to maximum serum (Tmax) values resulted from i.p. administration. In the peritoneal dissemination model, i.p. administration inhibited tumour growth and increased survival rate, whereas in the subcutaneous model, i.v. administration significantly inhibited tumour growth compared to i.p. administration. CONCLUSION: Administration of BK-UM by i.v. is both efficacious and safe.

Serum Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF) as a Biomarker for Primary Ovarian Cancer.

Ovarian cancer is the most lethal malignancy among gynaecological cancers. Although many anticancer agents have been developed for the treatment of ovarian cancer, it continues to have an extremely poor prognosis. Heparin-binding epidermal growth factor-like grown factor (HB-EGF) has been reported to be a rational therapeutic target for ovarian cancer. Here, we evaluated the clinical significance of serum HB-EGF by examining the association between prognosis and serum HB-EGF levels in patients with primary ovarian cancer. We found that high serum HB-EGF concentrations were significantly associated with poor prognosis in a combined cohort of patients with all stages of ovarian cancer, as well as in a subset of patients with advanced disease. In addition, serum HB-EGF levels increased as the cancer advanced. These data suggest that serum HB-EGF may be a target for the design of novel therapies for ovarian cancer.

BK-UM in patients with recurrent ovarian cancer or peritoneal cancer: a first-in-human phase-I study.

BACKGROUND: BK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration. METHODS: Eleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route. The dose was escalated (1.0, 2.0, 3.3, and 5.0 mg/m2) using a 3 + 3 design. RESULTS: Eight of 11 patients completed treatment. No dose-limiting toxicity (DLT) was experienced at dose levels 1 (1.0 mg/m2) and 2 (2.0 mg/m2). Grade 3 transient hypotension as an adverse event (defined as a DLT in the present study) was observed in two of four patients at dose level 3 (3.3 mg/m2). Treatment with BK-UM was associated with decreases in HB-EGF levels in serum and abdominal fluid in seven of 11 patients and five of eight patients, respectively. Clinical outcomes included a partial response in one patient, stable disease in five patients, and progressive disease in five patients. CONCLUSIONS: BK-UM was well tolerated at doses of 1.0 and 2.0 mg/m2, with evidence for clinical efficacy in patients with recurrent OC or PC. A dose of 2.0 mg/m2 BK-UM is recommended for subsequent clinical trials. TRIAL REGISTRATION: This trial was prospectively performed as an investigator-initiated clinical trial. The trial numbers are UMIN000001002 and UMIN000001001, with registration dates of 1/30/2008 and 2/4/2008, respectively. UMIN000001001 was registered as a trial for the continuous administration of BK-UM after UMIN000001002 .

MicroRNA-135a-3p as a promising biomarker and nucleic acid therapeutic agent for ovarian cancer.

Ovarian cancer is the most lethal gynecologic malignancy. Recently, several molecularly targeted anticancer agents have been developed for ovarian cancer; however, its prognosis remains extremely poor. The development of molecularly targeted therapy, as well as companion diagnostics, is required to improve outcomes for patients with ovarian cancer. In this study, to identify microRNAs (miRNAs) involved in the progression of ovarian cancer we analyzed serum miRNAs in patients with ovarian cancer using miRNA array and quantitative RT-PCR and examined the anticancer properties of miRNA expression in ovarian cancer cells. In patients with ovarian cancer, high amount of miR-135a-3p in serum samples was significantly associated with favorable clinical prognosis. The amount of miR-135a-3p was significantly decreased in patients with ovarian cancer compared with patients with ovarian cysts or normal ovaries. In SKOV-3 and ES-2 human ovarian cancer cells, enhanced expression of miR-135a-3p induced drug sensitivity to cisplatin and paclitaxel and suppressed cell proliferation and xenograft tumor growth. These findings suggest that miR-135a-3p may be considered as a biomarker and a therapeutic agent in ovarian cancer.