Proteinaceous microspheres for targeted RNA delivery prepared by an ultrasonic emulsification method

In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-delivery system. The preparation of RNA-loaded BSA spheres was accomplished using an environmental friendly method termed the “ultrasonic emulsification method”. It was demonstrated that ultrasonic waves do not cause the RNA chains to degrade and the RNA molecules remain untouched. The BSA–RNA complex was successfully introduced into mammalian (human) U2OS osteosarcoma cells and Trypanosoma brucei parasites. Using PVA coating of the RNA–BSA spheres we have achieved a significant increase in the number of microspheres penetrating mammalian cells. The mechanism of RNA encapsulation and the structure of the RNA–BSA complex are reported.Ulyana Shimanovich thanks Ministry of Science and Technology, Israel for the "Woman in Science" scholarship (3-8219)

Is Day-4 morula biopsy a feasible alternative for preimplantation genetic testing?

ObjectiveTo assess the efficacy and clinical outcome of PGT-M undertaken on Day-3, Day-4 and Day-4 "delayed" embryos that were unsuitable for biopsy on Day-3.Design and settingCohort-historical study of all consecutive patients admitted to the IVF-PGT-M program in a large tertiary center.Main outcome measure(s)The pregnancy rates and the percentages of complete, incomplete diagnosis, PCR failure, abnormal embryos in PGT of Day-3 cleavage-stage, Day-4 and Day-4 "delayed" embryos.Patients and methodsWe reviewed the medical files of all consecutive patients admitted to our IVF for a fresh IVF-PGT-M cycle. Patients were divided into 3 groups according to the day of blastomere biopsy: Day 3 cleavage-stage, Day-4 morula and Day-4 "delayed" embryos. The laboratory data, genetic diagnostic and clinical results were collected and compared between the different study groups.ResultsNine hundred and six patients underwent PGT-M cycles in our PGT program: 747, 127 and 32 in the Day-3, Day- 4 and Day-4 "delayed" groups, respectively. Ongoing pregnancy rates per transfer and per patient (15.8% and 9.4%, respectively) were non-significantly lower in the Day-4 "delayed", compared to Day-3 (21.4% and 17.5%, respectively) and Day-4 (24.3% and 19.7%, respectively). When comparing ALL morulas (Day-4 and Day-4 "delayed") to ALL cleavage-stage embryos (Day-3, Day-4 and Day-4 "delayed"), a significantly higher ongoing pregnancy rate was demonstrated following the transfer of embryos derived from morula biopsy, as compared to biopsy at the cleavage-stage (33.3% vs 20.5%, pConclusionDay-4 embryo biopsy is feasible and yields comparable and even higher ongoing pregnancy rate if undertaken at the morula stage. Further studies evaluating the cumulative live-birth rate per started cycles in Day-3 vs Day-4 embryo biopsy for PGT-M are warranted

Future fertility of patients with zero oocytes yield in their first IVF cycle attempt.

PurposeWe aim to estimate the future fertility of patient undergoing their first IVF cycle attempt with no oocyte retrieved, and to identify factors that might predict those who will conceive in subsequent IVF cycle attempt.MethodsA cohort retrospective study of all consecutive women attending our IVF unit, for their first IVF cycle attempt, between January 2013 to December 2019, who reached the ovum pick-up (OPU) stage with zero oocyte retrieved. Patients' characteristics and infertility-treatment-related variables in the first IVF cycle attempt were compared between those who conceived in a subsequent cycle and those who did not. Moreover, infertility-treatment-related variables during successful cycles resulting in pregnancy were compared to those without.Results59 met the study inclusion criteria, yielding zero oocytes. During the follow-up period, 12 (20.3%) women conceived (one conceived twice), and 8 (14%) gave birth to a live infant. Cumulative live-birth rate per OPU and per patients were 4% and 14%, respectively. Clinical pregnancies were achieved after 3.61+1.4 cycle attempts (range: 1-6), with no live-births following the fifth IVF cycle attempt. No in-between group differences were observed in ovarian stimulation variables of their first IVF cycle attempt. Moreover, in those cycles resulting in pregnancy, patients achieved a significantly higher number of fertilized oocytes (2.15+1.5 vs 0.94+1.5, respectively; pConclusionWomen yielding zero oocytes at their first IVF cycle attempt, may achieve 14% cumulative live-birth rate after 5 IVF cycle attempts. Moreover, those who conceived in subsequent IVF cycle attempts were those achieving 2 or more fertilized oocytes/TQE

The P Body Protein Dcp1a Is Hyper-phosphorylated during Mitosis

<div><p>Processing bodies (PBs) are non-membranous cytoplasmic structures found in all eukaryotes. Many of their components such as the Dcp1 and Dcp2 proteins are highly conserved. Using live-cell imaging we found that PB structures disassembled as cells prepared for cell division, and then began to reassemble during the late stages of cytokinesis. During the cell cycle and as cells passed through S phase, PB numbers increased. However, there was no memory of PB numbers between mother and daughter cells. Examination of hDcp1a and hDcp1b proteins by electrophoresis in mitotic cell extracts showed a pronounced slower migrating band, which was caused by hyper-phosphorylation of the protein. We found that hDcp1a is a phospho-protein during interphase that becomes hyper-phosphorylated in mitotic cells. Using truncations of hDcp1a we localized the region important for hyper-phosphorylation to the center of the protein. Mutational analysis demonstrated the importance of serine 315 in the hyper-phosphorylation process, while other serine residues tested had a minor affect. Live-cell imaging demonstrated that serine mutations in other regions of the protein affected the dynamics of hDcp1a association with the PB structure. Our work demonstrates the control of PB dynamics during the cell cycle via phosphorylation.</p> </div

Serine mutated Dcp1a proteins assembled into PBs.

<p>(A) Serine to alanine mutated GFP-Dcp1a proteins assembled in PBs in U2OS cells, and (B) disassembled during mitosis. Enlarged insets are boxed. DNA was counterstained with Hoechst. (Bar 20 µm).</p

Does daily co administration of gonadotropins and letrozole during the ovarian stimulation improve IVF outcome for poor and sub optimal responders?

Abstract Background Co-administration of letrozole during the first 5 days of ovarian stimulation was suggested to improve IVF outcomes in poor responders. We aimed to determine whether poor/sub-optimal responders might benefit from Letrozole co-treatment throughout the entire stimulation course. Methods We retrospectively reviewed the medical files of women who demonstrated poor (oocyte yield ≤3) and sub-optimal (4 ≤ oocyte yield ≤9) ovarian response during conventional multiple-dose antagonist stimulation protocols and were co-treated in a subsequent cycle with 5 mg Letrozole from the first day of stimulation until trigger day. A self-paired comparison between gonadotropins-only and gonadotropins-letrozole cycles was performed. Results Twenty-four patients were included. Mean patients’ age was 39.83 ± 4.60 and mean day-3-FSH was 12.77 ± 4.49 IU/m. Duration of stimulation and total gonadotropins dose were comparable between the two cycle groups. Peak estradiol levels were significantly lower in gonadotropins-letrozole cycles (2786.74 ± 2118.53 vs 1200.13 ± 535.98, p < 0.05). Number of retrieved oocytes (3.29 ± 2.15 vs 6.46 ± 3.20, p < 0.05), MII-oocytes (2.47 ± 1.65 vs 5.59 ± 3.20, p < 0.05), 2PN-embryos (1.78 ± 1.50, 4.04 ± 2.74, p < 0.05) and top-quality embryos (0.91 ± 0.97 vs. 2.35 ± 1.66, p < 0.05) were significantly higher in the gonadotropins-letrozole cycles. Clinical pregnancy rate in gonadotropins-letrozole cycles was 31.5%. Conclusion Letrozole co-treatment during the entire stimulation course improves ovarian response and IVF outcomes in poor/sub-optimal responders

Dcp1a is hyper-phosphorylated during cell division.

<p>Western blot analysis of (A) endogenous hDcp1a protein in U2OS cell extracts during interphase (untreated), metaphase (nocodazole block, Noc) and at G1/S (thymidine block, Thy), showed the appearance of slower migrating Dcp1a bands in metaphase cells. (B) Treatment of U2OS protein extracts from metaphase cells with a phosphatase (Noc+PPase) caused a reduction in the molecular weight of hDcp1a, compared to untreated, G1/S blocked (Thy), and metaphase blocked cells (Noc). This demonstrated that Dcp1a is hyper-phosphorylated during mitosis. Treatment with cycloheximide (Cyclo) for 1 or 4 hrs did not change the mobility of hDcp1a indicating that hyper-phosporylation is cell cycle dependent. (C) Shift in mobility due to hyper-phosphorylation in mitotic cells is seen using two different cell cycle blockers, nocodazole (Noc) and noscapine. Similarly, phosphatase treatment (Nos+PPase) caused a reduction in the molecular weight of Dcp1a from noscapine treated cells. Tubulin was used as a loading control.</p

Serine 315 is important for the hyper-phosphorylation of Dcp1a during cell division.

<p>Top - The S319A and S522,523A mutated GFP-Dcp1a proteins showed prominent hyper-phosphorylation patterns compared to the S315A protein. Bottom - blot comparing the mobility shifts of the mutated proteins from mitotic cell extracts showing that S315A is the least affected.</p

PB numbers increase as cells reach S/G2 phase of the cell cycle.

<p>(A) The Fucci markers mCherry-Cdt1 (red) and AmCyan1-Geminin (cyan) were expressed in U2OS cells and then cells were stained with an anti-Hedls (green) antibody to mark PBs. (Bar 20 µm). It was possible to detect the cell cycle phase using the intensity combination of the red and cyan markers in the cell, as explained in scheme below. (B) The number of PBs in each cell was counted and assigned a cell cycle phase according to the Fucci colors. The plot designates the average PB number in each phase (G1 n = 40, G1/S n = 15, S/G2 n = 40, M n = 10). Error bars represent STDEV and a T-Test was performed. (C) U2OS cells stably expressing GFP-Dcp1a were co-transfected with AmCyan1-Geminin and mCherry-Cdt1 and imaged for 15 hours. Frames show the cytoplasmic GFP-Dcp1a signal together with nuclear AmCyan1-Geminin staining that looks green due to the filter used. The plot represents the relative intensity analysis of all markers as quantified throughout the movie. Red – mean Cdt1 intensity, cyan – mean Geminin intensity, green – number of PBs.</p