Anin vitrothree-dimensional (3D) testicular organoid culture system for efficient gonocyte maintenance and propagation using frozen/thawed neonatal bovine testicular tissues.

Fertility preservation in prepubertal boys with cancer requires the cryopreservation of immature testicular tissues (ITTs) prior to gonadotoxic treatment. However, the limited number of germ cells in small human ITT biopsies necessitates the development of anin vitroculture system for germ cell expansion using frozen-thawed ITTs. Here, we generated testicular organoids for thein vitromaintenance and expansion of gonocytes from frozen-thawed two-week-old neonatal bovine ITTs. We investigated the effects of different cell-seeding densities, culture serums, seeding methods, and gonadotropin supplementations, on the maintenance and proliferation of enriched gonocytes. Our results demonstrated that enriched gonocytes and testicular cells from frozen-thawed neonatal ITTs could self-assemble into spheroid organoids in three days in an appropriate Matrigel-based culture environment. For the optimal formation of prepubertal testicular organoids, a seeding density of 1 × 106cells/well is recommended over other densities. This strategy results in organoids with a mean diameter of 60.53 ± 12.12 µm; the mean number of organoids was 5.57 ± 1.60/105µm2on day 11. The viability of organoids was maintained at 79.75 ± 2.99% after being frozen and thawed. Supplementing the culture medium with glial cell-derived neurotrophic factor, fibroblast growth factor 2, and leukemia inhibitory factor, increased the proportion of KI67-positive proliferating cells in organoids, elevated the expression ofC-KITbut reduced the expression ofGFRα1at day 28 when compared to those without hormone supplements(p< 0.05). In addition, supplementing the culture medium with follicle-stimulating hormone and testosterone helped to maintain a significantly higher viability (p< 0.05) in ITT organoids at day 28. These organoids could be cryopreserved for storage and thawed as needed. The successful generation of ITT organoids provides a valuable tool for establishingin vitrospermatogenesis, propagating human germ cells, investigating testicular physiology and the origin of germ cell tumors, and testing the toxicity of new drugs in future clinical applications.

Determining the optimal time interval between sample acquisition and cryopreservation when processing immature testicular tissue to preserve fertility.

The cryopreservation of immature testicular tissue (ITT) prior to gonadotoxic therapy is crucial for fertility preservation in prepubertal boys with cancer. However, the optimal holding time between tissue collection and cryopreservation has yet to be elucidated. Using the bovine model, we investigated four holding times (1, 6, 24, and 48 h) for ITTs before cryopreservation. Biopsies from two-week-old calves were stored in transport medium and cryopreserved following a standard slow-freezing clinical protocol. Thawed samples were then assessed for viability, morphology, and gene expression by haematoxylin and eosin (H&E) staining, immunohistochemistry and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Analysis failed to identify any significant changes in cell viability when compared between the different groups. Sertoli (Vimentin+) and proliferating cells (Ki67+) were well-preserved. The expression of genes related to germ cells, spermatogenesis (STRA8, PLZF, GFRα-1, C-KIT, THY1, UCHL-1, NANOG, OCT-4, CREM), and apoptosis (HSP70-2) remained stable over 48 h. However, seminiferous cord detachment increased significantly in the 48-h group (p < 0.05), with associated cord and SSC shrinkage. Collectively, our analyses indicate that bovine ITTs can be stored for up to 48 h prior to cryopreservation with no impact on cell viability and the expression levels of key genes. However, to preserve the morphology of frozen-thawed tissue, the ideal processing time would be within 24 h. Testicular tissues obtained from patients for fertility preservation often need to be transported over long distances to be cryopreserved in specialist centres. Our findings highlight the importance of determining optimal tissue transport times to ensure tissue quality in cryopreservation.

Dissociation, enrichment, and the in vitro formation of gonocyte colonies from cryopreserved neonatal bovine testicular tissues.

Gonocytes play an important role in early development of spermatogonial stem cells and fertility preservation to acquire more high quality gonocytes in vitro for further germ cell-related research and applications, it is necessarily needed to enrich and in vitro propagate gonocytes from cryopreserved bovine testicular tissues. This study aimed to investigate the isolation, enrichment, and colony formation of gonocytes in vitro for germ cell expansion from cryopreserved neonatal bovine testicular tissues. The effects of several different in vitro culture conditions, including seeding density, temperature, serum replacement and extracellular matrices were investigated for the maintenance, proliferation and formation of gonocyte colonies in vitro. Frozen/thawed two-week-old neonatal bovine testicular tissues were digested and gonocytes were enriched using a Percoll density gradient. Cell viability was accessed by trypan blue staining and cell apoptosis was evaluated by TUNEL assays. Gonocytes were identified and confirmed by immunofluorescence with the PGP9.5 germ cell marker and the OCT4 pluripotency marker while Sertoli cells were stained with vimentin. We found that neonatal bovine gonocytes were efficiently enriched by a 30%-40% Percoll density gradient (p < 0.05). No significant differences were detected between neonatal bovine testicular cells cultured at 34 °C or 37 °C. The formation of gonocyte colonies was observed in culture medium supplemented with knockout serum replacement (KSR), but not fetal bovine serum (FBS), at a seeding density higher than 5.0 × 104 cells/well. A greater number of gonocyte colonies were observed in culture plates coated with laminin (38.00 ± 6.24/well) and Matrigel (38.67 ± 3.78/well) when compared to plates coated with collagen IV and fibronectin (p < 0.05). In conclusion, bovine neonatal gonocytes were able to be efficiently isolated, enriched and maintained in gonocyte colonies in vitro; the development of this protocol provides vital information for the clinical translation of this technology and the future restoration of human fertility.

Management for delayed diagnosis in cesarean scar pregnancy with hemorrhage intra- or postuterine dilation and curettage.

AIM: This study aimed to examine the characteristics, management, and outcomes of delayed diagnosis of cesarean scar pregnancy (CSP) with hemorrhage intra- or postuterine curettage for early pregnancy termination. METHODS: The retrospective study, cases were identified from the interrogation of the hospital database and clinical data including the success rate of different treatments, vaginal bleeding time, abnormal beta-human chorionic gonadotropin (ß-hCG) time, and menstrual recovery time, preservation of uterus were analyzed. RESULTS: Medical records of 80 confirmed CSP cases with dilation and curettage (D&C) as primary treatment were analyzed; among them, 22 were treated with uterine arterial embolization (UAE) + methotrexate (MTX); 32 with UAE + surgery; 26 with only surgery or resection and repair. Treatment with UAE had less intraoperative blood loss (p < 0.05). UAE + surgery treatment had the highest success rate (96.8%, p < 0.05), the least vaginal bleeding duration after treatment (11.9 ± 9.6 days, p < 0.05), and least ß-hCG normalization time (17.4 ±  7.8 days, p < 0.05). CONCLUSION: UAE + surgery treatment is a favorable and effective option to control massive hemorrhage intra- or post-uterine curettage for early CSP termination.

Systematic bias between blinded independent central review and local assessment: literature review and analyses of 76 phase III randomised controlled trials in 45 688 patients with advanced solid tumour.

OBJECTIVE: Unbiased assessment of tumour response is crucial in randomised controlled trials (RCTs). Blinded independent central review is usually used as a supplemental or monitor to local assessment but is costly. The aim of this study is to investigate whether systematic bias existed in RCTs by comparing the treatment effects of efficacy endpoints between central and local assessments. DESIGN: Literature review, pooling analysis and correlation analysis. DATA SOURCES: PubMed, from 1 January 2010 to 30 June 2017. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Eligible articles are phase III RCTs comparing anticancer agents for advanced solid tumours. Additionally, the articles should report objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) or time to progression (TTP); the treatment effect of these endpoints, OR or HR, should be based on central and local assessments. RESULTS: Of 76 included trials involving 45 688 patients, 17 (22%) trials reported their endpoints with statistically inconsistent inferences (p value lower/higher than the probability of type I error) between central and local assessments; among them, 9 (53%) trials had statistically significant inference based on central assessment. Pooling analysis presented no systematic bias when comparing treatment effects of both assessments (ORR: OR=1.02 (95% CI 0.97 to 1.07), p=0.42, I2=0%; DCR: OR=0.97 (95% CI 0.92 to 1.03), p=0.32, I2=0%); PFS: HR=1.01 (95% CI 0.99 to 1.02), p=0.32, I2=0%; TTP: HR=1.04 (95% CI 0.95 to 1.14), p=0.37, I2=0%), regardless of funding source, mask, region, tumour type, study design, number of enrolled patients, response assessment criteria, primary endpoint and trials with statistically consistent/inconsistent inferences. Correlation analysis also presented no sign of systematic bias between central and local assessments (ORR, DCR, PFS: r>0.90, p<0.01; TTP: r=0.90, p=0.29). CONCLUSIONS: No systematic bias could be found between local and central assessments in phase III RCTs on solid tumours. However, statistically inconsistent inferences could be made in many trials between both assessments.

Early medical abortion with self-administered low-dose mifepristone in combination with misoprostol.

AIM: The aim of the present study was to investigate the safety and efficacy of low-dose mifepristone combined with self-administered misoprostol for termination of early pregnancy. METHODS: A total of 533 women seeking medical abortion in early pregnancy (≤49 days since the last menstrual period) were divided randomly into hospital- (H-Mis, 250) and self- (S-Mis, 283) administered misoprostol groups. Women in two groups took 100 mg of oral mifepristone in hospital followed by 200 µg of sublingual misoprostol 24 h later in hospital or home. The primary outcome parameter was complete abortion without surgical intervention. Secondary outcomes were uterine bleeding, return of regular menses, side effects and patient acceptability. RESULTS: High rates of complete abortion were observed for both the H-Mis group (243/250; 94.8%) and the S-Mis group (266/283; 94.0%). No significant differences in outcomes (complete abortion/failure rates) or side effects were observed between the two groups. General satisfaction rates were similar for the two groups (H-Mis, 231/250, 92.4%; S-Mis, 263/283, 92.9%; P > 0.05). Higher convenience of administration (H-Mis, 211/250, 84.4%; S-Mis, 270/283, 95.4%; P < 0.05) and privacy protection (H-Mis, 214/250, 85.6%; S-Mis, 267/283, 94.3%; P < 0.05) satisfaction rates were obtained for the S-Mis group than for the H-Mis group. CONCLUSION: Self-administered sublingual misoprostol is as safe and effective as hospital-administered misoprostol following low-dose mifepristone to terminate early pregnancy (≤49 days of amenorrhoea) with fewer side effects.

The alteration of T790M between 19 del and L858R in NSCLC in the course of EGFR-TKIs therapy: a literature-based pooled analysis.

BACKGROUND: Treatment-naive epidermal growth factor receptor (EGFR) T790M mutation is more inclined to coexist with L858R than with 19 del in non-small cell lung cancer (NSCLC) patients. However, EGFR-tyrosine kinase inhibitors (EGFR-TKIs) might alter this status. We sought to compare the prevalence of T790M upon acquired resistance to EGFR-TKIs between 19 del and L858R by assembling all existing data. METHODS: Electronic databases were comprehensively searched for eligible studies. The primary endpoint was the odds ratio (OR) of T790M mutation in NSCLC co-existing with L858R mutation and 19 del upon resistance to first-generation EGFR-TKIs. A random effects model was used. Stratified analysis was performed based on study type (retrospective and prospective), race (Asians and Caucasians) and sample type (tissue and plasma). RESULTS: A total of 25 studies involving 1,770 patients were included. The overall T790M existent rate was 45.25%. Post-resistance T790M was more frequent in 19 del than in L858R mutated patients (53% vs. 36%; OR 1.87; P<0.001). All outcomes of subgroup and overall analyses were similar. In contrast, we re-analyzed the previous meta-analysis, finding that the pooled rate of pretreatment T790M was 14% and 22% in 19 del and L858R respectively (OR 0.59; P<0.001). The increase of T790M rate was 2.79-fold in 19 del and only 0.63-fold in L858R in the course of EGFR-TKIs therapy. CONCLUSIONS: Opposite to the situation of de novo T790M, it was observed that T790M was more frequent in exon 19 deletion than in L858R among patients with acquired resistance to EGFR-TKIs. The difference in T790M alteration between 19 del and L858R encourages development of detection or treatment strategies for the specific resistance mechanism.

Robotic Versus Video-assisted Lobectomy/Segmentectomy for Lung Cancer: A Meta-analysis.

: Objective: To compare the safety/efficacy of the robotic-assisted lobectomy/segmentectomy (RAL/S) with the video-assisted lobectomy/segmentectomy (VAL/S) for radical lung cancer resection. BACKGROUND: It remains uncertain whether the newly developed RAL/S is comparable with the VAL/S. METHODS: A comprehensive search of online databases was performed. Perioperative outcomes were synthesized. Cumulative meta-analysis was performed to evaluate the temporal trend of pooled outcomes. Specific subgroups (propensity score matching studies, pure lobectomy studies) were examined. RESULTS: Analysis of 14 studies including a total of 7438 patients was performed. RAL/S was performed on 3239 patients, whereas the other 4199 patients underwent VAL/S. The 30-day mortality [0.7% vs 1.1%; odds ratio (OR) 0.53, P = 0.045] and conversion rate to open surgery (10.3% vs 11.9%; OR 0.57, P < 0.001) were significantly lower in patients who underwent RAL/S than VAL/S. Meanwhile, the postoperative complications (27.5% vs 28.2%; OR 0.95, P = 0.431), operation time [176.63 vs 162.74 min; standardized mean difference (SMD) 0.30, P = 0.086], duration of hospitalization (4.90 vs 5.23 days; SMD -0.08, P = 0.292), days to tube removal (4.10 vs 3.53 days; SMD 0.25, P = 0.120), retrieved lymph node (11.96 vs 10.67; SMD 0.46, P = 0.381), and retrieved lymph node station (4.98 vs 4.32; SMD 0.83, P = 0.261) were similar between the 2 groups. The cumulative meta-analyses suggested that the relative effects between 2 groups have already stabilized. All outcomes of subgroup and overall analyses were similar. CONCLUSIONS: This up-to-date meta-analysis confirms that RAL/S is a feasible and safe alternative to VAL/S for radical resection of lung cancer. Future studies should focus on the long-term benefits and cost effectiveness of RAL/S compared with VAL/S.

Evaluation bias in objective response rate and disease control rate between blinded independent central review and local assessment: a study-level pooled analysis of phase III randomized control trials in the past seven years.

BACKGROUND: In previous studies, complete-case implementation of blind independent central review has been considered unnecessary based on no sign of systematic bias between central and local assessments. In order to further evaluate its value, this study investigated evaluation status between both assessments in phase III trials of anti-cancer drugs for non-hematologic solid tumors. METHODS: Eligible trials were searched in PubMed with the date of Jan 1, 2010 to Jun 30, 2017. We compared objective response rate (ORR) and disease control rate (DCR) between central and local assessments by study-level pooled analysis and correlation analysis. In pooled analysis, direct comparison was measured by the odds ratio (OR) of central-assessed response status to local-assessed response status; to investigate evaluation bias between central and local assessments, the above calculated OR between experimental (exp-) and control (con-) arms were compared, measured by the ratio of OR. RESULTS: A total of 28 included trials involving 17,466 patients were included (28 with ORR, 16 with DCR). Pooled analysis showed central assessment reported lower ORR and DCR than local assessment, especially in trials with open-label design, central-assessed primary endpoint, and positive primary endpoint outcome, respectively. However, this finding could be found in both experimental [exp-ORR: OR=0.81 (95% CI: 0.76-0.87), P<0.01, I2=11%; exp-DCR: OR=0.90 (0.81-1.01), P=0.07, I2=42%] and control arms [con-ORR: OR=0.79 (0.72-0.85), P<0.01, I2=17%; con-DCR: OR=0.94 (0.86-1.02), P=0.14, I2=12%]. No sign of evaluation bias between two assessments was indicated through further analysis [ORR: ratio of OR=1.02 (0.97-1.07), P=0.42, I2=0%; DCR: ratio of OR=0.98 (0.93-1.03), P=0.37, I2=0%], regardless of mask (open/blind), sample size, tumor type, primary endpoint (central-assessed/local-assessed), and primary endpoint outcome (positive/negative). Correlation analysis demonstrated a high-degree concordance between central and local assessments (exp-ORR, con-ORR, exp-DCR, con-DCR: r>0.90, P<0.01). CONCLUSIONS: Blind independent central review remained irreplaceable to monitor local assessment, but its complete-case implementation may be unnecessary.

Efficacy, Safety, and Acceptability of Low-Dose Mifepristone and Self-Administered Misoprostol for Ultra-Early Medical Abortion: A Randomized Controlled Trial.

The aim of this study was to investigate the efficacy, safety, and acceptability of low-dose mifepristone combined with self-administered misoprostol for ultra-early medical abortion. A total of 744 women with ultra-early pregnancy (amenorrhea ≤35 days) who fulfilled the inclusion criteria were enrolled in the study. Equal numbers of participants were allocated randomly to the hospital administration and self-administration groups. All participants took 75 mg mifepristone at the initial visit and 400 µg oral misoprostol 24 hours later in the hospital or by self-administration. The primary end point was complete abortion. Secondary end points were rates of unscheduled reattendance, time required for and cost of hospital observation and follow-up, vaginal bleeding, adverse effects, menstrual disturbance in the posttreatment period, and satisfaction rating. No differences in the rates of complete abortion, unscheduled reattendance, vaginal bleeding, adverse effects, or return of posttreatment menstruation were observed. The time required for (and costs of) hospital observation and follow-up per participant was 557.82 minutes (and US$40.12) in the hospital administration group and 18.46 minutes (and US$1.96) in the self-administration group (both P < .001). Satisfaction rates were similar in both groups, but the rates of "very satisfied" responses (87.60% vs 25.41%) and follow-up compliance (loss to follow-up, 0.45% vs 7.70%) were higher in the self-administration group (both P < .001). Low-dose mifepristone combined with self-administered misoprostol for ultra-early pregnancy termination was as effective and safe as hospital administration, with greater acceptability and lower cost to the women.

Efficacy of Combined Laparoscopic and Hysteroscopic Repair of Post-Cesarean Section Uterine Diverticulum: A Retrospective Analysis.

BACKGROUND: Diverticulum, one of the long-term sequelae of cesarean section, can cause abnormal uterine bleeding and increase the risk of uterine scar rupture. In this study, we aimed to evaluate the efficacy of combined laparoscopic and hysteroscopic repair, a newly occurring method, treating post-cesarean section uterine scar diverticulum. METHODS: Data relating to 40 patients with post-cesarean section uterine diverticulum who underwent combined laparoscopic and hysteroscopic repair were retrospectively analyzed. Preoperative clinical manifestations, size of uterine defects, thickness of the lower uterine segment (LUS), and duration of menstruation were compared with follow-up findings at 1, 3, and 6 months after surgery. RESULTS: The average preoperative length and width of uterine diverticula and thickness of the lower uterine segment were recorded and analyzed. The average durations of menstruations at 1, 3, and 6 months after surgery were significantly shorter than the preoperative one (p < 0.05), respectively. At 6 months after surgery, the overall success improvement rate of surgery was 90% (36/40). Three patients (3/40 = 7.5%) developed partial improvement, and 1/40 (2.5%) was lost to follow-up. CONCLUSIONS: Our findings showed that combined treatment with laparoscopy and hysteroscopy was an effective method for the repair of post-cesarean section uterine diverticulum.