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Objective: To identify predictor microRNAs (miRNAs) from patients with repeated implantation failure (RIF). Design: Systemic analysis of miRNA profiles from the endometrium of patients undergoing in vitro fertilization (IVF). Setting: University research institute, private IVF center, and molecular testing laboratory. Patient(s): Twenty five infertile patients in the discovery cohort and 11 patients in the validation cohort. Interventions(s): None. Main Outcome Measure(s): A signature set of miRNA associated with the risk of RIF. Result(s): We designed a reproductive disease-related PanelChip to access endometrium miRNA profiles in patients undergoing IVF.Three major miRNA signatures, including hsa-miR-20b-5p, hsa-miR-155-5p, and hsa-miR-718, were identified using infinite combination signature search algorithm analysis from 25 patients in the discovery cohort undergoing IVF. These miRNAs were used as biomarkers in the validation cohort of 11 patients. Finally, the 3-miRNA signature was capable of predicting patients with RIF with an accuracy >90%. Conclusion(s):Our findings indicated that specific endometrial miRNAs can be applied as diagnostic biomarkers to predict RIF. Such information will definitely help to increase the success rate of implantation practice. (Fertil Steril Ò 2021;116:181-8. Ó2021 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo.

The genome of the white spot syndrome virus (WSSV) Taiwan isolate has many structural and non-structural genes that are arranged in clusters. Screening with Northern blots showed that at least four of these clusters produce polycistronic mRNA, and one of these (vp31/vp39b/vp11) was studied in detail. The vp31/vp39b/vp11 cluster produces two transcripts, including a large 3.4-kb polycistronic transcript of all three genes. No monocistronic vp39b mRNA was detected. TNT and in vitro translation assays showed that vp39b translation was independent of vp31 translation, and that ribosomal reinitiation was not a possible mechanism for vp39b. An unusually located IRES (internal ribosome entry site) element was identified in the vp31/vp39b coding region, and this region was able to promote the expression of a downstream firefly luciferase reporter. We show that vp31/vp39b/vp11 is representative of many other WSSV structural/non-structural gene clusters, and argue that these are also likely to produce polycistronic mRNAs and that use an IRES mechanism to regulate their translation.

Although shrimp white spot syndrome virus (WSSV) is a large double-stranded DNA virus (ϳ300 kbp), it expresses many polycistronic mRNAs that are likely to use internal ribosome entry site (IRES) elements for translation. A polycistronic mRNA encodes the gene of the highly expressed nonstructural protein ICP35, and here we use a dual-luciferase assay to demonstrate that this protein is translated cap independently by an IRES element located in the 5= untranslated region of icp35. A deletion analysis of this region showed that IRES activity was due to stem-loops VII and VIII. A promoterless assay, a reverse transcription-PCR together with quantitative real-time PCR analysis, and a stable stem-loop insertion upstream of the Renilla luciferase open reading frame were used, respectively, to rule out the possibility that cryptic promoter activity, abnormal splicing, or read-through was contributing to the IRES activity. In addition, a Northern blot analysis was used to confirm that only a single bicistronic mRNA was expressed. The importance of ICP35 to viral replication was demonstrated in a double-stranded RNA (dsRNA) interference knockdown experiment in which the mortality of the icp35 dsRNA group was significantly reduced. Tunicamycin was used to show that the ␣ subunit of eukaryotic initiation factor 2 is required for icp35 IRES activity. We also found that the intercalating drug quinacrine significantly inhibited icp35 IRES activity in vitro and reduced the mortality rate and viral copy number in WSSV-challenged shrimp. Lastly, in Sf9 insect cells, we found that knockdown of the gene for the Spodoptera frugiperda 40S ribosomal protein RPS10 decreased icp35 IRES-regulated firefly luciferase activity but had no effect on cap-dependent translation.