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.

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.

Diagnostic predictability of miR-4535 and miR-1915-5p expression in amniotic fluid for foetal morbidity of infection.

INTRODUCTION: Clinical prediction of foetal inflammatory response syndrome (FIRS) is highly necessary. We have previously reported that miR-4535 and miR-1915-5p are potential biomarkers for severe chorioamnionitis based on the results of microRNA array analysis. Therefore, we evaluated the relationship between foetal morbidity of infection and miR-4535, miR-1915-5p, interleukin (IL)-6, or 16S rDNA copy number levels in amniotic fluid from pregnant women with chorioamnionitis. METHODS: Amniotic fluid from 57 pregnant women with preterm premature membrane rupture or threatened premature labour were collected. Infants with WBC counts <5000/µL or >20,000/µL, CRP >0.5 mg/mL, or IgM >20 mg/mL at birth received a diagnosis of suspicious foetal infection, and those requiring antibiotic administration for >5 days were considered infected newborns. miR-4535, miR-1915-5p, and IL-6 levels and 16S rDNA copy number were evaluated. Mann-Whitney U test and Dunn's test were used for comparison. The area under the curve (AUC) and Youden index were calculated to examine the diagnostic accuracy of foetal morbidity of infection. RESULTS: miR-4535, miR-1915-5p, 16S rDNA, and IL-6 were significantly higher in patients with severe chorioamnionitis than in patients with chorionitis or sub-chorionitis (P < 0.05). miR-4535 and miR-1915-5p levels were significantly associated with WBC counts <5000/µL or >20,000/µL, CRP >0.5 mg/mL, or IgM >20 mg/mL (P < 0.05). AUC values of miR-4535 and miR-1915-5p indicated moderate or low accuracy for foetal morbidity of infection, while those of IL-6 and 16S rDNA seemed unreliable. DISCUSSION: MiR-4535 and miR-1915-5p levels in amniotic fluid may be considered clinically predictive for foetal morbidity of infection.

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.

Analysis of vaginal and endometrial microbiota communities in infertile women with a history of repeated implantation failure.

PURPOSE: To identify specific bacterial communities in vaginal and endometrial microbiotas as biomarkers of implantation failure by comprehensively analyzing their microbiotas using next-generation sequencing. METHODS: We investigated α- and ß-diversities of vaginal and endometrial microbiotas using 16S rRNA gene sequencing and compared their profiles between 145 women with repeated implantation failure (RIF) and 21 controls who lacked the factors responsible for implantation failure with a high probability of being healthy and fertile to identify specific bacteria that induce implantation failure. RESULTS: The endometrial microbiotas had higher α-diversities than did the vaginal microbiotas (P < .001). The microbiota profiles showed that vaginal and endometrial samples in RIF patients had significantly higher levels of 5 and 14 bacterial genera, respectively, than those in controls. Vaginal Lactobacillus rates in RIF patients were significantly lower at 76.4 ± 38.9% compared with those of the controls at 91.8 ± 22.7% (P = .018), but endometrial Lactobacillus rates did not significantly differ between the RIF patients and controls (56.2 ± 36.4% and 58.8 ± 37.0%, respectively, P = .79). CONCLUSIONS: Impaired microbiota communities containing specific bacteria in both the endometrium and vagina were associated with implantation failure. The vaginal Lactobacillus rates, but not the endometrial, may be a biomarker for RIF.

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.

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.

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.

Polymicrobial Amniotic Fluid Infection with Mycoplasma/Ureaplasma and Other Bacteria Induces Severe Intra-Amniotic Inflammation Associated with Poor Perinatal Prognosis in Preterm Labor.

PROBLEM: To study the relationship between perinatal prognosis in cases of preterm labor (PTL) and polymicrobial infection in amniotic fluid (AF) and intra-amniotic (IA) inflammation using a highly sensitive and reliable PCR-based method. METHOD OF STUDY: To detect prokaryotes using a nested PCR-based method, eukaryote-made thermostable DNA polymerase without bacterial DNA contamination was used in combination with bacterial universal primers. We collected AF aseptically from 118 PTL cases and 50 term subjects. RESULTS: The prevalence of microorganisms was 33% (39/118) by PCR and only 7.6% (9/118) by culture. PTL caused by a combination of positive Mycoplasma/Ureaplasma and other bacteria had significantly higher AF IL-8 levels and a significantly shorter amniocentesis-to-delivery interval. CONCLUSIONS: Our newly established PCR method is useful for detecting IA microorganisms. Polymicrobial infection with Mycoplasma/Ureaplasma and other bacteria induces severe IA inflammation associated with poor perinatal prognosis in PTL.

Predictive factors related to the efficacy of pelvic arterial embolization for postpartum hemorrhage: a retrospective analysis of 21 cases.

OBJECTIVE: This retrospective study aimed to determine the predictive factors for the efficacy of pelvic arterial embolization for postpartum hemorrhage. MATERIALS AND METHODS: Twenty-one patients who underwent pelvic arterial embolization for postpartum hemorrhage of >1000 mL between September 2006 and September 2011 were enrolled in this study. The patients were divided into two subgroups according to the blood loss and time from the end of pelvic arterial embolization to complete hemostasis: good-response (16 patients) and poor-response groups (5 patients). The following predictive factors were compared between the groups: (1) patient characteristics; (2) blood loss; (3) time between delivery (or onset of bleeding) and pelvic arterial embolization; (4) obstetrical disseminated intravascular coagulation score comprising clinical background, clinical signs, and laboratory data; (5) individual disseminated intravascular coagulation score; (6) shock index; and (7) laboratory data including platelet count, prothrombin time-international normalized ratio, fibrinogen, fibrin degradation products, and antithrombin-III at the time of pelvic arterial embolization. RESULTS: In the poor-response group, the obstetrical and individual disseminated intravascular coagulation scores and prothrombin time-international normalized ratio were higher than those in the good-response group (p < 0.05). Platelet count, fibrinogen, and fibrin degradation products were lower than those in the good-response group (p < 0.05). All obstetrical disseminated intravascular coagulation scores in the poor-response group were >9 points. CONCLUSION: The efficacy of pelvic arterial embolization is related to the presence or absence of coagulation disorders. When the obstetrical disseminated intravascular coagulation score is high (>9 points), the efficacy may be poor.

The successful detection of postpartum unruptured vaginal pseudoaneurysm using ultrasonography: a case report.

INTRODUCTION: We report the first case of the successful detection of postpartum unruptured vaginal pseudoaneurysm using power- and pulsed-Doppler ultrasonography after delivery. CASE DESCRIPTION: A 38-year-old primiparous Japanese woman had a vaginal laceration with pulsatile bleeding after delivering by vacuum extraction. Transvaginal ultrasonography of the repaired vaginal wall showed an 18 × 20 mm hematoma within which a 6 × 7 mm pulsating anechoic mass was detected. Power-Doppler ultrasonography showed blood flow signals in the anechoic mass. Arterial waveforms detected in pulsed-Doppler mode were suggestive of unruptured pseudoaneurysm. Careful monitoring with contrast-enhanced computed tomography showed an increase in the size of the pseudoaneurysm on the fourth postpartum day. On the sixth postpartum day, massive vaginal bleeding occurred. Emergency angiography revealed strong staining with extravasation from the left vaginal artery, confirming the diagnosis of pseudoaneurysm. Embolization for hemostasis was successfully performed. DISCUSSION AND EVALUATION: As far as we know, our case is the first in which an unruptured vaginal pseudoaneurysm was diagnosed using ultrasonography. The differential diagnoses of pseudoaneurysm are arteriovenous malformations including arteriovenous fistula. This case had the typical ultrasonographic patterns of pseudoaneurysm in which the presence of one or two cystic masses in B-mode and color- and/or power-Doppler flow signals was demonstrated along with high-resistance arterial flow waveforms in pulsed-Doppler mode. Sequential examinations of contrast-enhanced CT showed ongoing development of the pseudoaneurysm. In retrospect, we could have performed angiography for embolization when the unruptured pseudoaneurysm was diagnosed, or at the latest when ongoing development of the pseudo-aneurysm was recognized, irrespective of whether symptoms were present. CONCLUSIONS: Ultrasonography is a non-invasive and clinically useful modality in the differential diagnosis of pseudoaneurysm. Contrast-enhanced computed tomography with or without ultrasonography can be useful for sequential monitoring of the size of unruptured pseudoaneurysms.