Metronomic capecitabine with rapamycin exerts an immunosuppressive effect by inducing ferroptosis of CD4+ T cells after liver transplantation in rat.

Liver transplantation is one of the most effective treatments for hepatocellular carcinoma (HCC). The balance between inhibiting immune rejection and preventing tumor recurrence after liver transplantation is the key to determining the long-term prognosis of patients with HCC after liver transplantation. In our previous study, we found that capecitabine (CAP), an effective drug for the treatment of HCC, could exert an immunosuppressive effect after liver transplantation by inducing T cell ferroptosis. Recent studies have shown that ferroptosis is highly associated with autophagy. In this study, we confirmed that the autophagy inducer rapamycin (RAPA) combined with metronomic capecitabine (mCAP) inhibits glutathione peroxidase 4 (GPX4) and promotes ferroptosis in CD4+ T cells to exert immunosuppressive effects after rat liver transplantation. Compared with RAPA or mCAP alone, the combination of RAPA and mCAP could adequately reduce liver injury in rats with acute rejection after transplantation. The CD4+ T cell counts in peripheral blood, spleen, and transplanted liver of recipient rats significantly decreased, and the oxidative stress level and ferrous ion concentration of CD4+ T cells significantly increased in the combination group. In vitro, the combination of drugs significantly promoted autophagy, decreased GPX4 protein expression, and induced ferroptosis in CD4+ T cells. In conclusion, the autophagy inducer RAPA improved the mCAP-induced ferroptosis in CD4+ T cells. Our results support the concept of ferroptosis as an autophagy-dependent cell death and suggest that the combination of ferroptosis inducers and autophagy inducers is a new research direction for improving immunosuppressive regimens after liver transplantation.

Effects of NGS-based PGT-a for idiopathic recurrent pregnancy loss and implantation failure: a retrospective cohort study.

To clarify the effect of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) combined with trophectoderm (TE) biopsy on the pregnancy outcomes of idiopathic recurrent pregnancy loss (iRPL) and idiopathic recurrent implantation failure (iRIF), we conducted a retrospective cohort study of 212 iRPL couples and 66 iRIF couples who underwent PGT-A or conventional in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment. The implantation rate (IR) per transfer (64.2%), clinical pregnancy rate (CPR) per transfer (57.5%), and live birth rate (LBR) per transfer (45%) of iRPL couples of the PGT-A treatment group were significantly higher (p < 0.05) than those of the conventional IVF/ICSI group (IR per transfer,38.2%; CPR per transfer,33.3%; LBR per transfer, 28.4%), whereas the pregnancy loss rate (PLR) per transfer was similar between the two groups. These effects were also significant (p < 0.05) in iRPL couples with advanced maternal age (AMA, ≥35 years), whereas no significant differences were found in clinical outcomes between the PGT-A and conventional IVF/ICSI groups in younger iRPL couples (<35 years). The cumulative clinical outcomes of iRPL couples were comparable between the PGT-A and conventional IVF/ICSI groups. No significant differences were found in any clinical outcomes between the PGT-A and conventional IVF/ICSI groups for young or AMA couples with iRIF. In conclusion, NGS-based PGT-A involving TE biopsy may be useful for iRPL women to shorten the time to pregnancy and reduce their physical and psychological burden, especially for iRPL women with AMA; however, couples with iRIF may not benefit from PGT-A treatment. Considering the small sample size of the iRIF group, further investigations with a larger sample size are needed to verify our findings.

Ultrasound-Guided Human Islet Transplantation: Safety, Feasibility, and Efficacy Analysis.

RATIONALE AND OBJECTIVES: We aimed to analyze the safety, feasibility, and efficacy of human islet transplantation (IT) using ultrasound (US) throughout the entire procedure. MATERIALS AND METHODS: A total of 22 recipients (18 males; mean age 42.6 ± 17.5years) with 35 procedures were retrospective included. Under US guidance, percutaneous transhepatic portal catheterization was successfully performed through a right-sided transhepatic approach, and islets were infused into the main portal vein. Color Doppler and contrast-enhanced ultrasound were used to guide the procedure and monitor the complications. After infusion of the islet mass, the access track was embolized by embolic material. If hemorrhage persisted, US-guided radiofrequency ablation (RFA) was performed to stop bleeding. Factors that could affect the complication were analyzed. After transplantation, primary graft function was evaluated with a ß-score 1month after the last islet infusion. RESULTS: The technical success rates were 100% with a single puncture attempt. Six (17.1%) abdominal bleeding episodes were immediately stopped by US-guided RFA. No portal vein thrombosis were encountered. Dialysis (OR (Odd Ratio): 32.0; 95% CI: 1.561-656.054; and P = .025) was identified as a significant factor associated with bleeding. Primary graft function was optimal in eight patients (36.4%), suboptimal in 13 patients (59.1%), and poor in one patient (4.5%). CONCLUSION: In conclusion, whole-procedure US-guided IT is a safe, feasible, and effective method for diabetes. Complications are either self-limiting or manageable with noninvasive treatment.

Generation of a human induced pluripotent stem cell line (SYSUTFi001-A) from infiltrating cytotoxic T cells in hepatocellular carcinoma (HCC).

In this study, we report a novel induced pluripotent stem cell (iPSC) line SYSUTFi001-A derived from cytotoxic T cells (CTLs) infiltrating in hepatocellular carcinoma (HCC), using an integrative Sendai virus vector. This pluripotent cell line shows a normal karyotype and can be redifferentiated to the rejuvenated CTLs targeted to HCC. The cell line SYSUTFi001-A can be further used to perform vitro and vivo anti-tumor assays and design future cell replacement therapies.

Integrative network analysis of circular RNAs reveals regulatory mechanisms for hepatic specification of human iPSC-derived endoderm.

BACKGROUND: Human-induced pluripotent stem cell (hiPSC)-derived functional hepatic endoderm (HE) is supposed to be an alternative option for replacement therapy for end-stage liver disease. However, the high heterogeneity of HE cell populations is still challenging. Hepatic specification of definitive endoderm (DE) is an essential stage for HE induction in vitro. Recent studies have suggested that circular RNAs (circRNAs) determine the fate of stem cells by acting as competing endogenous RNAs (ceRNAs). To date, the relationships between endogenous circRNAs and hepatic specification remain elusive. METHODS: The identities of DE and HE derived from hiPSCs were determined by qPCR, cell immunofluorescence, and ELISA. Differentially expressed circRNAs (DEcircRNAs) were analysed using the Arraystar Human circRNA Array. qPCR was performed to validate the candidate DEcircRNAs. Intersecting differentially expressed genes (DEGs) of the GSE128060 and GSE66282 data sets and the DEcircRNA-predicted mRNAs were imported into Cytoscape for ceRNA networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were involved in the enrichment analysis. Hepatic markers and Wnt/ß-catenin were detected in hsa_circ_004658-overexpressing cells by western blotting. Dual-luciferase reporter assays were used to evaluate the direct binding among hsa_circ_004658, miRNA-1200 and CDX2. DE cells were transfected with miR-1200 mimics, adenovirus containing CDX2, and Wnt/ß-catenin was detected by western blotting. RESULTS: hiPSC-derived DE and HE were obtained at 4 and 9 days after differentiation, as determined by hepatic markers. During hepatic specification, 626 upregulated and 208 downregulated DEcircRNAs were identified. Nine candidate DEcircRNAs were validated by qPCR. In the ceRNA networks, 111 circRNA-miRNA-mRNA pairs were involved, including 90 pairs associated with hsa_circ_004658. In addition, 53 DEGs were identified among the intersecting mRNAs of the GSE128060 and GSE66282 data sets and the hsa_circ_004658-targeted mRNAs. KEGG and GO analyses showed that the DEGs associated with hsa_circ_004658 were mainly enriched in the WNT signalling pathway. Furthermore, hsa_circ_004658 was preliminarily verified to promote hepatic specification, as determined by hepatic markers (AFP, ALB, HNF4A, and CK19) (p < 0.05). This promotive effect may be related to the inhibition of the Wnt/ß-catenin signalling pathway (detected by ß-catenin, p-ß-catenin, and TCF4) when hsa_circ_004658 was overexpressed (p < 0.05). Dual-luciferase reporter assays showed that there were binding sites for miR-1200 in the hsa_circ_004658 sequence, and confirmed the candidate DEG (CDX2) as a miR-1200 target. The level of miR-1200 decreased and the level of CDX2 protein expression increased when hsa_circ_004658 was overexpressed (p < 0.05). In addition, the results showed that CDX2 may suppress the Wnt/ß-catenin signalling during hepatic specification (p < 0.05). CONCLUSIONS: This study analysed the profiles of circRNAs during hepatic specification. We identified the hsa_circ_004658/miR-1200/CDX2 axis and preliminarily verified its effect on the Wnt/ß-catenin signalling pathway during hepatic specification. These results provide novel insight into the molecular mechanisms involved in hepatic specification and could improve liver development in the future.

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform.

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

Research Progress on the Microregulatory Mechanisms of Fertilization: A Review.

The process of fertilization includes sperm capacitation, hyperactivation, an acrosome reaction and the release of acrosome enzymes, membrane fusion and channel formation, the release of the sperm nucleus, and gamete fusion. This process is closely related to the shape and vitality of the sperm, acrosome enzyme release, and the zona pellucida structure of the egg, as well as the opening and closing of various ion (e.g., calcium) channels, the regulation of signaling pathways such as cyclic adenosine monophosphate-protein kinase A, the release of progesterone, and the coupling of G-proteins. The interaction among multiple factors and their precise regulation give rise to multiple cascading regulatory processes. Problems with any factor will affect the success rate of fertilization. Recent studies have shown that with rapid societal development, the incidence of male infertility is increasing and occurs at younger ages. According to World Health Organization statistics, 15% of couples of childbearing ages have infertility problems, of which 50% are caused by male factors. Additionally, the cause of infertility cannot be identified in as many as 60% to 75% of male infertility patients. In this article, we review the research progress on the microregulation of fertilization and mechanisms underlying this process to identify causes and develop novel prevention and treatment strategies for male infertility.

PGT-A: The biology and hidden failures of randomized control trials.

OBJECTIVE: Preimplantation Genetic Testing - Aneuploidy (PGT-A) for embryo selection has undergone significant advancements in the last 2 decades and yet many studies still fail to demonstrate any clinical benefits over traditional embryo morphology selection (Mo-S). To understand this conundrum, we performed a multi-center clinical study of PGT-A patients, where Mo-S and euploid selection (Eu-S) outcomes were directly compared. METHOD: All suitable blastocysts were biopsied and analyzed for chromosome copy number. Outcomes (positive beta hCG, implantation, ongoing pregnancy, and live birth rates) for Eu-S were compared to Mo-S using single embryo transfers. RESULTS: Compared to Eu-S embryos, Mo-S embryos resulted in significant reduction of outcomes for positive beta hCG (p = 0.0005), implantation (p = 0.0008), ongoing pregnancy (p = 0.0046), livebirth (p = 0.0112), babies per transfer (p = 0.0112), and babies per embryo transferred (p = 0.0112). Morphology selection resulted in patients of all age groups having non-euploid embryos chosen for transfer. Post-hoc evaluation of individual clinic performances showed variable transfer outcomes that could potentially confound the true benefits of PGT-A. CONCLUSION: Embryo chromosome status is central to improved embryo transfer outcomes and sole reliance on current morphology-based selection practices, without Eu-S, will always compromise outcomes. Often overlooked but a major effector of successful PGT-A outcomes are individual clinic performances.

Luteal phase support for natural cycle frozen embryo transfer: a meta-analysis.

PURPOSE: We aimed to evaluate the effect of luteal phase support (LPS) on pregnancy outcome in natural cycle frozen embryo transfer (NC-FET). METHOD: We searched PubMed, Cochrane Library, Embase for related literature from start to February 2020. Relative risk ratio (RR) and 95% confidence intervals (95% CI) in random-effects, fixed-effects models were calculated using Review Manager 5.3. RESULTS: Totally 9 studies were included in the meta-analysis. The results showed no significant difference could be found regarding chemical pregnancy rate (RR 1.07, 95% CI 0.93-1.22; I2 = 54%) and miscarriage rate (RR 0.92, 95% CI 0.70-1.22; I2 = 0%) between the LPS groups and no LPS groups in NC-FET. LPS groups has increased the rate of clinical pregnancy rate (RR 1.23, 95% CI 1.12-1.34; I2 = 52%) compared with no LPS groups. Subgroup analysis according to trigger administration also showed a significant difference between the two groups. CONCLUSION: LPS might improve the clinical pregnancy rate in NC-FET. HCG trigger for ovulating may result in luteal phase deficiency. LPS subsequently improved clinical pregnancy rate and chemical pregnancy rate for patients undergoing HCG trigger and NC-FET. RETROSPECTIVELY REGISTERED: This meta-analysis was registered at PROSPERO, PROSPERO ID is CRD42020171758.

The predictive role of preoperative serum glutamate dehydrogenase levels in microvascular invasion and hepatocellular carcinoma prognosis following liver transplantation-a single center retrospective study.

BACKGROUND: As a critical metabolic substrate, glutamine is not only involved in the progression of many cancers but is also related to angiogenesis. Glutamate dehydrogenase (GLDH), a key enzyme in glutamine metabolism, has been reported to regulate tumor proliferation; however, its relationship with microvascular invasion (MVI) is unclear. This study evaluated the ability of preoperative serum GLDH levels to predict MVI and the long-term survival of hepatocellular carcinoma (HCC) patients after liver transplantation (LT). METHODS: HCC patients that underwent LT from January 2015 to May 2020 at the First Affiliated Hospital of Sun Yat-Sen University were enrolled in our retrospective analysis. Clinicopathological variables were extracted from medical records. A receiver operating characteristic curve was created to determine the optimal cut-off value of GLDH for MVI. RESULTS: Preoperative GLDH was significantly elevated in the MVI-positive group (U = 454.00, p = 0.000). The optimal cut-off value of GLDH for MVI was 7.45 U/L, with an area under the curve of 0.747 (95% CI [0.639-0.856], p = 0.000). The sensitivity was 79.3%, while the specificity was 64.5%. GLDH > 7.45 U/L (p = 0.023) and maximum diameter >5 cm (p = 0.001) were independent risk factors for the presence of MVI. Patients with GLDH > 7.45 U/L had significantly poorer overall survival (p = 0.001) and recurrence-free survival (p = 0.001) after LT than patients with GLDH ≤ 7.45 U/L. Similarly, patients with MVI were associated with poor survival (p = 0.000). CONCLUSIONS: Preoperative elevated serum GLDH levels predict MVI and poorer long-term survival for HCC after LT.

IVF embryo choices and pregnancy outcomes.

OBJECTIVE: Investigate the chromosome status and transfer outcomes of embryos selected using routine "best morphology" IVF practices. METHOD: A prospective multi-center, non-selection cohort study involving patients undertaking IVF treatment. Study entry conditions were blastocyst biopsy, >1 embryo with chromosome analysis and frozen transfer of the best morphology embryo. Primary analyses were ßhCG positive, implantation, ongoing pregnancy and birth rates and pregnancy-stage progression failures. RESULTS: After transfer, embryo chromosome status was assigned and outcomes divided into two primary groups - euploids (n = 135) and aneuploids (n = 53). Compared to euploid embryo transfers, aneuploid embryos had significantly lower primary outcomes (+ßhCG: 67% vs. 30%, p < 0.0001; IR: 56% vs. 19%, p < 0.0001; ongoing week 12: 51% vs. 9%, p < 0.0001; and livebirths: 50% vs. 8%, p < 0.0001, respectively). Transfers were further subdivided into smaller groups according to their main chromosomal feature. Stage analysis showed higher failure rates for aneuploids to initiate a pregnancy (p < 0.0001), higher subclinical miscarriage rate (p = 0.0402) and higher clinical miscarriage rate (p = 0.0038). CONCLUSION: Routine morphology-based embryo selection resulted in a high euploid selection rate but a significant number of aneuploid embryos were still inadvertently selected for transfer (28%) with the subsequent high failure rates for pregnancy initiation and progression having implications for appropriate patient management.

Diabetes Mellitus Causes Male Reproductive Dysfunction: A Review of the Evidence and Mechanisms.

The metabolic disorders caused by diabetes can lead to various complications, including dysfunction of the male reproductive system. In patients with diabetes, long-term hyperglycemia results in diabetic vascular neuropathy, oxidative stress injury, abnormal zinc metabolism, and insulin resistance syndrome. In addition, insulin deficiency and resistance in diabetes can damage the hypothalamus, pituitary gland, gonads, and perigonads. This can reduce the secretion of sex hormones including gonadotropin-releasing hormone, follicle stimulating hormone, luteinizing hormone, and testosterone, and can lead to testicular atrophy, stromal cell atrophy, seminiferous tubule damage, spermatogenic cell damage, and other structural injuries of the male reproductive organs. These actions can affect male fertility and reproductive health. Herein, we review studies that report a causative role of diabetes in male reproductive function. We also discuss the evidence-based mechanisms involved in the processes of diabetes-related male sexual and reproductive dysfunction as well as the progress in treatment.

Novel mutations in ZP1 and ZP2 cause primary infertility due to empty follicle syndrome and abnormal zona pellucida.

PURPOSE: Mutations in the zona pellucida glycoprotein genes have been reported to be associated with empty follicle syndrome (EFS) and abnormal zona pellucida (ZP). In this study, we performed genetic analysis in the patients with female infertility due to abnormal zona pellucida and empty follicle syndrome to identify the disease-causing gene mutations in these patients. METHODS: We characterized three patients from two independent families who had suffered from empty follicle syndrome or abnormal zona pellucida. Whole exome sequencing and Sanger sequencing were used to identify the mutations in the families. Western blot was used to check the expression of wild type and mutant disease genes. RESULTS: We identified two novel mutations in these patients, including a novel compound heterozygous mutation (c.507delC, p. His170fs; c.239 G>A, p. Cys80Tyr and c.241 T>C, p. Tyr81His) in ZP1 gene and a compound mutation in ZP2 gene (c.860_861delTG, p.Val287fs and c.1924 C>T, p.Arg642Ter). Expression of the mutant ZP1 protein (p. Cys80Tyr and p. Tyr81His) is significantly decreased compared with the wild-type ZP1. Other three mutations produce truncated proteins. CONCLUSIONS: Our findings expand the mutational spectrum of ZP1 and ZP2 genes associated with EFS and abnormal oocytes and provide new support for the genetic diagnosis of female infertility.

Perinatal outcomes after vitrified-warmed day 5 blastocyst transfers compared to vitrified-warmed day 6 blastocyst transfers: A meta analysis.

AIMS: The aim of this meta-analysis was to compare the perinatal outcomes between the vitrified-warmed day 5 blastocyst transfer (BT) and the vitrified-warmed day 6 blastocyst transfer (BT). METHODS: PubMed, EMBASE and the Cochrane Library were searched for the perinatal outcomes after in vitro fertilisation / intracytoplasmic sperm injection (IVF/ICSI) from inception to October 2018.The perinatal outcomes included birth weight, gestational age, number of males, premature delivery, birth defects, and neonatal deaths. We used a random effect model to analyse the summary risk ratios (RRs) and mean difference (WMD) with 95 % confidence intervals (CIs). RESULTS: Eight retrospective studies that met the inclusion criteria were included. Compared with vitrified-warmed day 5 BT, vitrified-warmed day 6 BT was associated with increased birth weight (WMD = -80.39; 95 % CI = -151.8 to -8.97; I2 = 41 %, P = 0.03);There was no significant difference in gestational age (WMD = 0.10; 95 % CI =-0.07-0.27; I2 = 0%, P = 0.24), number of males (RR 0.93, 95 % CI 0.78-1.10; I2 = 43 %), premature delivery (RR 0.84, 95 % CI 0.13-5.27; I2 = 72 %), birth defects (RR 1.48, 95 % CI 0.71-3.11; I2 = 0%) and neonatal deaths (RR 1.2, 95 % CI 0.25-5.71; I2 = 0%) between the two groups. CONCLUSIONS: Vitrified-warmed day 6 BT is associated with increased birth weight rather than day 5 BT. There was no difference in gestational age, number of males, premature deliveries, birth defects, and neonatal death, between the two groups. These results concluded that vitrified-warmed day 6 BT has no difference compared with vitrified-warmed day 5 BT in regard to adverse impact on perinatal outcomes.

Novel homozygous mutations in PATL2 lead to female infertility with oocyte maturation arrest.

PURPOSE: To identify the disease gene in 40 patients with female infertility due to oocyte maturation arrest. METHODS: Genomic DNA was extracted from peripheral blood of 40 patients and their family members. Whole-exome sequencing was performed on the patients, and the PATL2 mutations were identified and confirmed by Sanger sequencing. Harmfulness of the mutations was analyzed by SIFT, Polyphen-2, Mutation Taster, and M-CAP software, and we used western immunoblotting analysis to check the effect of mutations on PATL2 protein expression in vitro. RESULTS: Two novel missense mutations c.1528C>A (p.Pro510Thr) and c.1376C>A (p.Ser459Tyr) in PATL2 were identified in three patients (7.5%) from two consanguineous families in our cohort. We found that mutations in PATL2 resulted in variable oocyte phenotypes, including GV arrest, MI arrest, and morphologic abnormalities. Western immunoblotting analysis showed that the expression levels of the two novel mutant PATL2 proteins decreased significantly. CONCLUSIONS: We identified two novel PATL2 mutations that caused oocyte maturation arrest and abnormal morphology, and variable phenotypes in patients.

Association of antinuclear antibody with clinical outcome of patients undergoing in vitro fertilization/intracytoplasmic sperm injection treatment: A meta-analysis.

PROBLEM: Several recent studies have investigated the relationship between antinuclear antibodies (ANAs) and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes. This meta-analysis evaluated the effect of ANA on clinical outcome for patients undergoing IVF/ICSI treatment. METHOD OF STUDY: A systemic survey of the literature was performed using PubMed, EMBASE and the Cochrane Library databases, through January 2019. Evaluated outcomes included clinical pregnancy rate (CPR), miscarriage rate (MR) and implantation rate (IR). Relative risk ratio (RR) and 95% confidence intervals (95% CI) were used for dichotomous data. Meta-analyses were performed with Review Manager 5.3 software. RESULTS: Eleven eligible studies were found. For IVF/ICSI cycles, compared with an ANA(-) group, infertile patients with ANA(+) had significantly reduced incidence of CPR (RR 0.66, 95% CI 0.56-0.79; I2  = 60%) and IR (RR 0.61, 95% CI 0.49-0.76; I2  = 75%), and had a higher MR (RR 1.81, 95% CI 1.40-2.36; I2  = 48%). CONCLUSION: This study suggested that ANA might cause poor pregnancy outcomes for infertile women undergoing IVF/ICSI treatment.

Exposure to podophyllotoxin inhibits oocyte meiosis by disturbing meiotic spindle formation.

Podophyllotoxin is used as medical cream which is widely applied to genital warts and molluscum contagiosum. Although previous study showed that podophyllotoxin had minimal toxicity, it was forbidden to use during pregnancy since it might be toxic to the embryos. In present study we used mouse as the model and tried to examine whether podophyllotoxin exposure was toxic to oocyte maturation, which further affected embryo development. Our results showed that podophyllotoxin exposure inhibited mouse oocyte maturation, showing with the failure of polar body extrusion, and the inhibitory effects of podophyllotoxin on oocytes was dose-depended. Further studies showed that the meiotic spindle formation was disturbed, the chromosomes were misaligned and the fluorescence signal of microtubule was decreased, indicating that podophyllotoxin may affect microtubule dynamics for spindle organization. Moreover, the oocytes which reached metaphase II under podophyllotoxin exposure also showed aberrant spindle morphology and chromosome misalignment, and the embryos generated from these oocytes showed low developmental competence. We also found that the localization of p44/42 MAPK and gamma-tubulin was disrupted, which further confirmed the effects of podophyllotoxin on meiotic spindle formation. In all, our results indicated that podophyllotoxin exposure could affect mouse oocyte maturation by disturbing microtubule dynamics and meiotic spindle formation.

Clinical application of next-generation sequencing in preimplantation genetic diagnosis cycles for Robertsonian and reciprocal translocations.

PURPOSE: The purpose of this study was to apply next-generation sequencing (NGS) technology to identify chromosomally normal embryos for transfer in preimplantation genetic diagnosis (PGD) cycles for translocations. METHODS: A total of 21 translocation couples with a history of infertility and repeated miscarriage presented at our PGD clinic for 24-chromosome embryo testing using copy number variation sequencing (CNV-Seq). RESULTS: Testing of 98 embryo samples identified 68 aneuploid (69.4 %) and 30 (30.6 %) euploid embryos. Among the aneuploid embryos, the most common abnormalities were segmental translocation imbalances, followed by whole autosomal trisomies and monosomies, segmental imbalances of non-translocation chromosomes, and mosaicism. In all unbalanced embryos resulting from reciprocal translocations, CNV-Seq precisely identified both segmental imbalances, extending from the predicted breakpoints to the chromosome termini. From the 21 PGD cycles, eight patients had all abnormal embryos and 13 patients had at least one normal/balanced and euploid embryo available for transfer. In nine intrauterine transfer cycles, seven healthy babies have been born. In four of the seven children tested at 18 weeks gestation, the karyotypes matched with the original PGD results. CONCLUSION: In clinical PGD translocation cycles, CNV-Seq displayed the hallmarks of a comprehensive diagnostic technology for high-resolution 24-chromosome testing of embryos, capable of identifying true euploid embryos for transfer.

[Recurrent implantation failure: gamete and embryo factors].

Despite the immense achievement in the field of IVF in recent years, many patients still suffer from recurrent implantation failure. Therefore, much attention has been drawn to its etiology and treatment. Chromosomal abnormality, sperm DNA damage, zona hardening, and inappropriate culture conditions are important factors that lead to recurrent implantation failure. Results of studies suggest that preimplantation genetic screening does not improve the rate of implantation or live birth. Comparative genomic hybridization array and single nucleotide polymorphism array could offer a more comprehensive screening of chromosomes. Assisted hatching may help to solve the problem of zona hardening in some situations. Co-culture and blastocyst transfer could be conducive to the improvement of the rates of implantation and pregnancy. Cytoplasmic transfer may give a solution to ooplasmic composition anomalies.

[Comparison of Alarelin and Triptorelin in the long-protocol ovulation induction in in vitro fertilization and embryo transfer].

OBJECTIVE: To compare the pituitary down-regulatory effects of the two gonadotropin-releasing hormone agonists Alarelin and Triptorelin in the long protocol of ovulation induction in in vitro fertilization and embryo transfer (IVF-ET). METHODS: We included in this study 122 patients aged 24-39 years treated by IVF-ET for secondary infertility, with 10-20 pre-antral follicles and obstruction of the fallopian tube. Seventy-eight of them received Alarelin, and the other 44 Triptorelin. Comparative analyses were made on the pituitary down-regulatory effects of the two gonadotropin-releasing hormone agonists and the clinical outcomes of IVF-ET. RESULTS: No premature LH surge and ovulation, nor severe hyperovarian stimulation syndrome was found in either group. There were no significant differences between the two groups in the mean dose and duration of gonodatropin treatment, the numbers of oocytes retrieved, mature oocytes and top-quality embryos, and the rates of 2PN, multi-sperm fertilization, cleavage, embryo transfer, embryo implantation, clinical pregnancy and early miscarriage (P > 0.05), but the rate of cancelled cycles was significantly higher in the Triptorelin than in the Alarelin group (P < 0.05). CONCLUSION: Alarelin and Triptorelin can achieve similar pituitary down-regulatory effects and clinical outcomes in IVF-ET when used in the long protocol of ovulation induction.