Sperm Selection for ICSI: Do We Have a Winner?

In assisted reproductive technology (ART), the aim of sperm cells’ preparation is to select competent spermatozoa with the highest fertilization potential and in this context, the intracytoplasmic sperm injection (ICSI) represents the most applied technique for fertilization. This makes the process of identifying the perfect spermatozoa extremely important. A number of methods have now been developed to mimic some of the natural selection processes that exist in the female reproductive tract. Although many studies have been conducted to identify the election technique, many doubts and disagreements still remain. In this review, we will discuss all the sperm cell selection techniques currently available for ICSI, starting from the most basic methodologies and continuing with those techniques suitable for sperm cells with reduced motility. Furthermore, different techniques that exploit some sperm membrane characteristics and the most advanced strategy for sperm selection based on microfluidics, will be examined. Finally, a new sperm selection method based on a micro swim-up directly on the ICSI dish will be analyzed. Eventually, advantages and disadvantages of each technique will be debated, trying to draw reasonable conclusions on their efficacy in order to establish the gold standard method.

Increased B Cell Selection Stringency In Germinal Centers Can Explain Improved COVID-19 Vaccine Efficacies With Low Dose Prime or Delayed Boost

The efficacy of COVID-19 vaccines appears to depend in complex ways on the vaccine dosage and the interval between the prime and boost doses. Unexpectedly, lower dose prime and longer prime-boost intervals have yielded higher efficacies in clinical trials. To elucidate the origins of these effects, we developed a stochastic simulation model of the germinal center (GC) reaction and predicted the antibody responses elicited by different vaccination protocols. The simulations predicted that a lower dose prime could increase the selection stringency in GCs due to reduced antigen availability, resulting in the selection of GC B cells with higher affinities for the target antigen. The boost could relax this selection stringency and allow the expansion of the higher affinity GC B cells selected, improving the overall response. With a longer dosing interval, the decay in the antigen with time following the prime could further increase the selection stringency, amplifying this effect. The effect remained in our simulations even when new GCs following the boost had to be seeded by memory B cells formed following the prime. These predictions offer a plausible explanation of the observed paradoxical effects of dosage and dosing interval on vaccine efficacy. Tuning the selection stringency in the GCs using prime-boost dosages and dosing intervals as handles may help improve vaccine efficacies.

Safety and Preliminary GvHD Mitigation in First in Human Clinical Trial of Apograft in Match Related Stem Cell Transplantation (SCT)

Graft versus host disease (GvHD) is a major frequent adverse event (AE) and the primary cause of morbidity and mortality following allogeneic stem cell transplantation (ASCT). Numerous attempts to reduce incidence and severity of GvHD using a variety of cell selection methods have been employed, but they all suffer from a risk/benefit trade-off where reduction of GvHD-causing cells leads to reduced engraftment and/or reduced graft vs. tumor (GvT) effect. We have previously reported development of a new method for cell selection, which take advantage of cells' differential sensitivity to CD95 receptor mediated apoptosis. This technology enables the differential selection of transplant related immune-toxicity cells (e.g., TH1, TH17, terminally differentiated B cells and antigen presenting cells {APCs}) while maintaining stem and progenitor cells. Thus, selecting the right mix of cells may enable both GvHD reduction and maintenance of the graft's effectiveness. Here we report results of our first in humans clinical trial using this functional cell selection technology (named ApoGraft). This study was an Open-Label Phase 1/2 Pilot, Staggered Four-Cohort Safety and Proof-of-Concept Study of ApoGraft in the Prevention of Acute Graft Versus Host Disease in Match related SCT (NCT02828878). The primary objective of the trial was to demonstrate safety and tolerability of ApoGraft in adult patients with hemato-oncology disorders. The key secondary objective was engraftment and prevention of acute GvHD. Eleven patients were enrolled into this study in 4 sequential cohorts of 3 patients each, except for 2 patients in cohort 4. Enrolled subjects' transplant indications included AML (n=7), ALL (n=1), MDS (n=2) and biphenotypic acute leukemia (n=1). ApoGraft was manufactured ex-vivo from donor mobilized peripheral blood cells (MPBCs) collected via a single apheresis donation that was incubated for 2 hours with FasL protein (10, 25, 50 and 100 ng/ml per cohort), and washed before transplantation to the recipient. Patients were administered with a single ApoGraft treatment. The ApoGraft product median CD34+ yield was 72%, with a median of 4.3x10^6 CD34+ cells/Kg/patient. All patients received GVHD prophylaxis with short-course methotrexate and Cyclosporin A. No AEs or serious AEs (SAEs) were recorded during stem cell infusion. Six months follow-up showed ApoGraft transplantation to be well tolerated with no related AEs or SAEs. Successful engraftment was observed in all subjects within the expected timeframe. Median time to neutrophils or platelets engraftment was 14 days. Lower incidence of grade 2-4 aGvHD was observed in Cohorts 3 and 4 (n=2) as compared to Cohorts 1 and 2 (n=5). None of the higher FasL dose Cohort 3 and Cohort 4 patients had grade 3 and 4 aGvHD compared to 3 patients in cohorts 1 and 2. Over 97% of bone marrow at 28 days post-transplant were of donor origin, indicating full donor chimerism, except for the two MDS patients; one had shown full donor chimerism, which was reduced at day 180 (55%). The other had a donor chimerism of 92% at day 28. None of the patients relapsed during the study period. Four of 6 patients in the 10 and 25 ng/ml cohorts had 7 episodes of CMV viremia, as compared with 1 episode in the 50ng/ml cohort and none in the 100ng/ml cohort. Ten patients survived and were progression-free by study day 180. One MDS patient died during the study due to study unrelated secondary graft failure. Our results show evidence that the ApoGraft product was well tolerated at all FasL doses and demonstrated preliminary signs of GvHD mitigation when the higher ex-vivo doses of FasL (50 and 100 ng/ml) were used. Disclosures Zuckerman: AbbVie: Honoraria; Orgenesis Inc.: Honoraria; Janssen: Honoraria; BioSight Ltd: Honoraria; Novartis: Honoraria; Gilead Sciences: Honoraria, Speakers Bureau; Cellect Biotechnology: Honoraria. Yehudai-Ofir: Novartis: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees. Kamar: Cellect Biotheraputics: Consultancy.

Mouse oocytes develop in cysts with the help of nurse cells

Mammalian oocytes develop initially in cysts containing many more germ cells than the primordial oocytes they generate. We identified abundant nurse cells with reduced unique molecular identifiers (UMI)/cell from ovaries aged E14.5 to P1. Low UMI nurse cells are found in cysts and express the same major meiotic genes as pro-oocytes of the same stage, suggesting they are oocyte sisters that are signaled to transfer cytoplasm at different times and only subsequently diverge. Oocyte vs nurse cell selection occurs in cysts with a robust microtubule cytoskeleton, that closely interact with somatic cells and that develop a dense actin cytoskeleton around nurse cell nuclei that are held back from cytoplasmic transfer. Mouse and Drosophila nurse cells undergo programmed cell death by acidification from adjacent somatic pre-granulosa cells that express V-ATPases and cathepsin proteins. Disrupting acidification in cultured mouse ovaries blocked nurse cell turnover. About 200 genes are induced in mouse dictyate oocytes as previously reported, including Tuba1c and Tubb2b, genes that we find contribute to Balbiani body formation. Thus, mouse oocytes are specified within germline cysts and develop with the assistance of nurse cells using highly conserved mechanisms.

Specialized transendothelial dendritic cells mediate thymic T-cell selection against blood-borne macromolecules

T cells undergo rigorous selection in the thymus to ensure self-tolerance and prevent autoimmunity, with this process requiring innocuous self-antigens (Ags) to be presented to thymocytes. Self-Ags are either expressed by thymic stroma cells or transported to the thymus from the periphery by migratory dendritic cells (DCs); meanwhile, small blood-borne peptides can access the thymic parenchyma by diffusing across the vascular lining. Here we describe an additional pathway of thymic Ag acquisition that enables circulating antigenic macromolecules to access both murine and human thymi. This pathway depends on a subset of thymus-resident DCs, distinct from both parenchymal and circulating migratory DCs, that are positioned in immediate proximity to thymic microvessels where they extend cellular processes across the endothelial barrier into the blood stream. Transendothelial positioning of DCs depends on DC-expressed CX3CR1 and its endothelial ligand, CX3CL1, and disrupting this chemokine pathway prevents thymic acquisition of circulating proteins and compromises negative selection of Ag-reactive thymocytes. Thus, transendothelial DCs represent a mechanism by which the thymus can actively acquire blood-borne Ags to induce and maintain central tolerance.