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Objective: To assess whether co-culture with vitrified-warmed cumulus cells (CCs) in media drops improves rescue in vitro maturation (IVM) of previously vitrified immature oocytes. Previous studies have shown improved rescue IVM of fresh immature oocytes when cocultured with CCs in a three-dimensional matrix. However, the scheduling and workload of embryologists would benefit from a simpler IVM approach, particularly in the setting of time-sensitive oncofertility oocyte cryopreservation (OC) cases. Although the yield of developmentally competent mature metaphase II (MII) oocytes is increased when rescue IVM is performed before cryopreservation, it is unknown whether maturation of previously vitrified immature oocytes is improved after coculture with CCs in a simple system not involving a three-dimensional matrix. Design: Randomized controlled trial. Setting: Academic hospital. Patients: A total of 320 (160 germinal vesicles [GVs] and 160 metaphase I [MI]) immature oocytes and autologous CC clumps were vitrified from patients who were undergoing planned OC or intracytoplasmic sperm injection from July 2020 until September 2021. Interventions: On warming, the oocytes were randomized to culture in IVM media with CCs (+CC) or without CCs (-CC). Germinal vesicles and MI oocytes were cultured in 25 μL (SAGE IVM medium) for 32 hours and 20-22 hours, respectively. Main outcome measures: Oocytes with a polar body (MII) were randomized to confocal microscopy for analysis of spindle integrity and chromosomal alignment to assess nuclear maturity or to parthenogenetic activation to assess cytoplasmic maturity. Wilcoxon rank sum tests for continuous variables and the chi square or Fisher's exact test for categorical variables assessed statistical significance. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated. Results: Patient demographic characteristics were similar for both the GV and MI groups after randomization to +CC vs. -CC. No statistically significant differences were observed between +CC vs. -CC groups regarding the percentage of MII from either GV (42.5% [34/80] vs. 52.5% [42/80]; RR 0.81; 95% CI: 0.57-1.15]) or MI (76.3% [61/80]; vs. 72.5% [58/80]; RR 1.05; 95% CI: 0.88-1.26]) oocytes. An increased percentage of GV-matured MIIs underwent parthenogenetic activation in the +CC group (92.3% [12/13] vs. 70.8% [17/24]), but the difference was not statistically significant (RR 1.30; 95% CI: 0.97-1.75), whereas the activation rate was identical for MI-matured oocytes (74.3% [26/35] vs. 75.0% [18/24], CC+ vs. CC-; RR 0.99; 95% CI: 0.74-1.32). No significant differences were observed between +CC vs. -CC groups for cleavage of parthenotes from GV-matured oocytes (91.7% [11/12] vs. 82.4% [14/17]) or blastulation (0 for both) or for MI-matured oocytes (cleavage: 80.8% [21/26] vs. 94.4% [17/18]; blastulation: 0 [0/26] vs. 16.7% [3/18]). Further, no significant differences were observed between +CC vs. -CC for GV-matured oocytes regarding incidence of bipolar spindles (38.9% [7/18] vs. 33.3% [5/15]) or aligned chromosomes (22.2% [4/18] vs. 0.0 [0/15]); or for MI-matured oocytes (bipolar spindle: 38.9% [7/18] vs. 42.9% [2/28]); aligned chromosomes (35.3% [6/17] vs. 24.1% [7/29]). Conclusions: Cumulus cell co-culture in this simple two-dimensional system does not improve rescue IVM of vitrified, warmed immature oocytes, at least by the markers assessed here. Further work is required to assess the efficacy of this system given its potential to provide flexibility in a busy, in vitro fertilization clinic. 

The generation of neurons in the central nervous system is a complex, stepwise process necessitating the coordinated activity of mitotic progenitors known as radial glia. Following neural tube closure, radial glia undergo a period of active proliferation to rapidly expand their population, creating a densely packed neurepithelium. Simultaneously, radial glia positioned across the neural tube are uniquely specified to produce diverse neuronal sub-types. Although these cellular dynamics are well studied, the molecular mechanisms governing them are poorly understood. The six-transmembrane Glycerophosphodiester Phosphodiesterase proteins (GDE2, GDE3, and GDE6) comprise a family of cell-surface enzymes expressed in the embryonic nervous system. GDE proteins can release Glycosylphosphatidylinositol-anchored proteins from the cell surfaceviacleavage of their lipid anchor. GDE2 has established roles in motor neuron differentiation and oligodendrocyte maturation, and GDE3 regulates oligodendrocyte precursor cell proliferation. Here, we describe a role for GDE6 in early neural tube development. Using RNAscope, we show thatGde6mRNA is expressed by ventricular zone progenitors in the caudal neural tube. Utilizing in-ovo electroporation, we show that GDE6 overexpression promotes neural tube hyperplasia and ectopic growths of the neurepithelium. At later stages, electroporated embryos exhibit an expansion of the ventral patterning domains accompanied by reduced cross-repression. Ultimately, electroporated embryos fail to produce the full complement of post-mitotic motor neurons. Our findings indicate that GDE6 overexpression significantly affects radial glia function and positions GDE6 as a complementary factor to GDE2 during neurogenesis. 

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the viability of upper and lower motor neurons. Current options for treatment are limited, necessitating deeper understanding of the mechanisms underlying ALS pathogenesis. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that acts on the cell surface to cleave the glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane. GDE2 is required for the survival of spinal motor neurons but whether GDE2 neuroprotective activity is disrupted in ALS is not known. We utilized a combination of mouse models and patient post-mortem samples to evaluate GDE2 functionality in ALS. Haplogenetic reduction of GDE2 exacerbated motor neuron degeneration and loss in SOD1 G93A mice but not in control SOD1 WT transgenic animals, indicating that GDE2 neuroprotective function is diminished in the context of SOD1 G93A . In tissue samples from patients with ALS, total levels of GDE2 protein were equivalent to healthy controls; however, membrane levels of GDE2 were substantially reduced. Indeed, GDE2 was found to aberrantly accumulate in intracellular compartments of ALS motor cortex, consistent with a disruption of GDE2 function at the cell surface. Supporting the impairment of GDE2 activity in ALS, tandem-mass-tag mass spectrometry revealed a pronounced reduction of GPI-anchored proteins released into the CSF of patients with ALS compared with control patients. Taken together, this study provides cellular and biochemical evidence that GDE2 distribution and activity is disrupted in ALS, supporting the notion that the failure of GDE2-dependent neuroprotective pathways contributes to neurodegeneration and motor neuron loss in disease. These observations highlight the dysregulation of GPI-anchored protein pathways as candidate mediators of disease onset and progression and accordingly, provide new insight into the mechanisms underlying ALS pathogenesis.