[Silencing circular RNA_ embryonic-lethal abnormal vision-like protein 2(circELAVL2) inhibits the proliferation and induces apoptosis of mouse testicular TM4 Sertoli cells].

Objective To investigate the effects of silencing circular RNA_embryonic-lethal abnormal vision-like protein 2 (circELAVL2, mmu_circ_0011854) on the proliferation and apoptosis of mouse testicular TM4 Sertoli cells and identify its underlying mechanisms. Methods Small interfering RNAs (siRNA) were used to silence the expression of circELAVL2 in TM4 cells. CCK-8 assay and 5-ethynyl-2'- deoxyuridine (EdU) assay were used to detect the effects of circELAVL2 on the proliferation of TM4 cells. Flow cytometry and TdT-mediated dUTP nick-end labeling (TUNEL) were used to detect the effects of circELAVL2 on the apoptosis of TM4 cells. Bioinformatics were adopted to predict the potential binding sites between circEALVL2 and miR-382-3p and luciferase reporter assays to detect the binding abilities between circELAVL2 and miR-382-3p. Real time-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of circELAVL2 and miR-382-3p. Results After silencing the expression of circELAVL2, the proliferating rate and the proportion of EdU positive TM4 cells were decreased, while apoptotic levels of TM4 cells were increased. Mechanism study found circELAVL2 could bind with miR-382-3p and inhibit its expression. Conclusion circELAVL2 promotes cell proliferation and suppresses cell apoptosis of mouse TM4 Sertoli cells by binding with and inhibiting miR-382-3p.

A Modified Vessel-sparing Microsurgical Vasoepididymostomy.

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, preserving the vasal vessels during MVE might better simulate the normal physiological structure and be meaningful for patients who have undergone varicocelectomy. Nevertheless, preserving the vasal vessels might elevate the risk of increasing the tension of anastomosis, affecting the patency rate and leading to delayed postoperative bleeding. Therefore, we developed a novel vessel-sparing MVE to make it safer. Here is a summary of the improvements to the procedure. 1) The retrograde dissociation of the vasal vessels on the proximal testicular side was adopted as the main method, and the anterograde dissociation of the vasal vessels on the distal testicular side was adopted as a supplement to dissociate the vasal vessels to be preserved. This improvement ensures the blood supply to the vas deferens that will be used for anastomosis and also provides longer vasal vessels, which reduces the tension of anastomosis. 2) By fixing the vas deferens to be anastomosed and the broken end of the vas deferens, the free vasal vessels get fixed, which resolves the problem of transmission of vas tension to the vasal vessels and reduces the risk of vasal vessel hemorrhage. 3) Dissociation of the vas deferens after opening the tunica vaginalis increases the mobilization of the vas deferens, which also makes the new procedure easier to complete. The evaluation of the outcomes of this new procedure showed that no significant postoperative complications occurred in the patients, and the patency rate was no different from that of the conventional procedure. Therefore, this new, improved procedure can be considered safe, with satisfactory postoperative results.

Clinical application of NGS-based SNP haplotyping for PGT-M of methylmalonic acidemia.

This study describes a successful case of preimplantation genetic testing for the monogenic disease (PGT-M) of methylmalonic acidemia (MMA). To avoid the transmission of pathogenic mutations and unnecessary pregnancy termination we applied next-generation sequencing (NGS)-based haplotyping on a couple with a previously deceased MMA offspring. After embryo preparation, all samples were amplified successfully by whole genome amplification. We performed preimplantation genetic testing for aneuploidy (PGT-A) to determine the copy number of embryos' chromosomes. PGT-A results showed five blastocysts (2, 11, 14, 15 and 16) with balanced chromosomes (46, XN). Two techniques were used for PGT-M. Sanger sequencing was used to detect the mutations of MMUT gene directly, and NGS-based single nucleotide polymorphism (SNP) haplotyping was used to distinguish the chromosomes that carried the mutation. Sanger sequencing and NGS-based SNP haplotyping confirmed that samples 2 and 15 carried c.730insTT, samples 11 and 15 carried c.1105 C > T and samples 14 and 16 did not carry any mutation. Thus, blastocyst 14 was transferred into the mother's uterus. After prenatal diagnosis at 18 weeks of gestation, a healthy infant without MMUT mutation was born at full term. This study highlights the efficiency of NGS-based SNP haplotyping for PGT-M of MMA.Abbreviations: MMA: methylmalonic acidemia; MMUT: methylmalonyl-CoA mutase; PGT-M: preimplantation genetic testing for monogenic disease; PGD: preimplantation genetic diagnosis; IVF: in vitro fertilization; ADO: allele dropout; WGA: whole genome amplification; SNP: single nucleotide polymorphism; NGS: next-generation sequencing; PND: prenatal diagnosis; ICSI: intracytoplasmic sperm injection; TE: trophectoderm; DOP-PCR: degenerate oligonucleotide primed polymerase chain reaction; PGT-A: preimplantation genetic testing for aneuploidy; PCR: polymerase chain reaction.

Clinical application of NGS-based SNP haplotyping for the preimplantation genetic diagnosis of primary open angle glaucoma.

The current study describes a successful case of preimplantation genetic diagnosis (PGD) of primary open angle glaucoma (POAG) and verifies the efficiency of next-generation sequencing (NGS)-based haplotyping for PGD of POAG. In this study, we applied NGS as part of PGD to effectively detect POAG prior to embryo implantation and avoid the prospect of pregnancy termination in event of vertical inheritance of POAG. We used the technique of multiple annealing and looping based amplification cycles (MALBAC) to conduct whole genome amplification (WGA) and to reduce the allele dropout (ADO). We also employed Sanger sequencing to directly detect the mutation c.1109 C > T in MYOC and NGS-based single nucleotide polymorphism (SNP) haplotyping to distinguish the chromosomes that carried the mutation. Copy number variation (CNV) analysis was carried out to determine the copy number of embryos' chromosomes. Of the 4 blastocysts obtained in this study, only 2 (sample 5 and 7) could be successfully amplified by WGA. CNV results indicated that chromosomes of both these samples were balanced (46, XN). Sanger sequencing and NGS-based SNP haplotyping confirmed that sample 7 carried the mutation c.1109 C > T in MYOC, while sample 5 did not. Moreover, no ADO was observed. Thus, blastocyst 5 was transferred into the uterus of the patient, and a healthy baby without the MYOC mutation c. 1109C>T was born 39 weeks after transplantation. Our study suggests that NGS-based SNP haplotyping is an effective technique for the PGD of POAG. Abbreviations: PGD: preimplantation genetic diagnosis; POAG: primary open angle glaucoma; NGS: next-generation sequencing; MALBAC: multiple annealing and looping based amplification cycles chemistry; WGA: whole genome amplification; ADO: allele dropout; SNP: single nucleotide polymorphism; CNV: copy number variation; MYOC: Myocilin; OPTN: Optineurin; WDR36: WD repeat domain 36; CYP1B1: Cytochrome P450 1 B Chain; ICSI: intracytoplasmic sperm injection; TFNA: testicular fine-needle aspiration; TE: trophectoderm; PCR: polymerase chain reaction.