In vitro maturation of human oocytes maintaining good development potential for rescue intracytoplasmic sperm injection with fresh sperm.

BACKGROUND: The outcomes of the use of commercial in vitro maturation (IVM) medium to culture immature oocytes obtained from conventional ovulation induction, followed by rescue intracytoplasmic sperm injection (RICSI), are not ideal. It is thus difficult to widely adopt this approach in clinical practice. Therefore, it is necessary to explore methods for improving the clinical outcome of IVM. AIM: To study the effect of sperm on the developmental potential of in vitro-matured oocytes in conventional culture. METHODS: This was a retrospective study of patients whose immature oocytes were harvested from conventional oocyte stimulation cycles and underwent ICSI at our hospital between June 2018 and August 2020. RICSI was performed using sperm collected on the day of oocyte harvest (old) and sperm collected on the day of RICSI (fresh) and oocytes matured in vitro after 24 h of culture in conventional medium. The rates of in vitro oocyte maturation, normal fertilization, normal cleavage, day-3 top-quality embryos, and useful blastocyst formation were compared between the two groups. RESULTS: In total, 102 germinal vesicle (GV)-stage immature oocytes were cultured in the old sperm group. In the fresh sperm group, 122 GV-stage immature oocytes were collected and cultured in vitro for 24 h. There were no significant differences in the general conditions of males and females between the two groups (P > 0.05). The oocyte maturation, normal fertilization, and normal cleavage rates of the old and fresh groups were 51.0% vs 55.7%, 61.5% vs 64.7%, and 93.8% vs 93.2%, respectively. None of the rates differed significantly (P > 0.05) between the two groups. However, the day-3 top-quality embryo and useful blastocyst rates of the old and fresh sperm groups were 16.6% vs 63.4%; 6.67% vs 34.6%, respectively. The day-3 top-quality embryos and useful blastocyst rates of the old sperm group were significantly lower than those of the fresh group (P < 0.05). CONCLUSION: In vitro maturation with conventional culture medium combined with the use of fresh sperm collected on the day of RICSI is an easy-to-implement strategy for patients whose oocytes are completely or mostly immature.

[Preimplantation Genetic Diagnosis of α/ß Complex Thalassemia by Next Generation Sequencing].

OBJECTIVE: To explore the application value of next generation sequencing (NGS) in preimplantation genetic diagnosis of α/ß complex thalassemia couple. METHODS: The coding regions of α-globin genes (HBA1, HBA2) and ß-globin gene (HBB) were selected as the target regions. The high-density and closely linked single nucleotide polymorphism (SNP) sites were selected as the genetic linkage markers in the upstream and downstream 2M regions of the gene. After NGS, the effective SNP sites were selected to construct the haplotype of the couple, and the risk chromosome of the mutation carried by the couple was determined. The NGS technology was used to sequence the variations of HBA1, HBA2 and HBB directly and construct haplotype linkage analysis for preimplantation genetic diagnosis. RESULTS: Direct sequencing and haplotype linkage analysis of HBA1, HBA2 and HBB showed that two of the six blastocysts were α/ß complex thalassemia, one was ß-thalassemia heterozygote, two were α-thalassemias heterozygotes, and one was intermediate α-thalassemia. A well-developed embryo underwent preimplantation genetic diagnosis was implanted into the mother's uterus, and a healthy infant was born at term. CONCLUSION: Preimplantation genetic diagnosis can be carried out by NGS technology in α/ß complex thalassemia couples, and abortion caused by aneuploid embryo selection can be avoided.

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae.

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors. Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described. This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

A recombinant AeDNA containing the insect-specific toxin, BmK IT1, displayed an increasing pathogenicity on Aedes albopictus.

The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT(50) and LD(50) bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.

[Effect of cinobufagin on nuclear factor-kappaB pathway in HepG2 cells].

OBJECTIVE: To investigate the effect of cinobufagin on nuclear factor-kappaB (NF-kappaB) pathway of liver cancer cell line HepG2. METHODS: Dual-luciferase cis-reporting system was used to detect the relative value of pNF-kappaB-TA-luc upon tumor necrosis factor-alpha (TNF-alpha) stimulation of NF-kappaB pathway. Western blotting was used to detect the protein level of NF-kappaB p65, and RT-PCR was used to detect the gene transcription level of intercellular adhesion molecule-1 (ICAM-1), a target downstream gene of NF-kappaB. RESULTS: At the concentration of 0.25 and 0.5 microg/ml, cinobufagin significantly lowered the relative value of luciferase (P<0.05). The results of Western blotting showed that cinobufagin significantly suppressed the protein expression of NF-kappaB p65. The transcription level of ICAM-1 was reduced by different doses of cinobufagin. CONCLUSION: The anti-cancer effect of cinobufagin may be related to its activity in inhibiting the activation of NF-kappaB pathway.

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

OBJECTIVE: To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). METHODS: Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. RESULTS: MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. CONCLUSION: The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.