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Background: Many clinical trials have reported the use of mesenchymal stromal cells (MSCs) following the indication of severe SARS-CoV-2 infection. However, in the COVID19 pandemic context, academic laboratories had to adapt a production process to obtain MSCs in a very short time. Production processes, especially freezing/thawing cycles, or culture medium have impacts on MSC properties. We evaluated the impact of an intermediate cryopreservation state during MSC culture to increase production yields. Methods: Seven Wharton’s jelly (WJ)-MSC batches generated from seven different umbilical cords with only one cryopreservation step and 13 WJ-MSC batches produced with intermediate freezing were formed according to good manufacturing practices. The identity (phenotype and clonogenic capacities), safety (karyotype, telomerase activity, sterility, and donor qualification), and functionality (viability, mixed lymphocyte reaction) were analyzed. Results: No significant differences between MSC production processes were observed, except for the clonogenic capacity, which was decreased, although it always remained above our specifications. Conclusions: Intermediate cryopreservation allows an increase in the production yield and has little impact on the basic characteristics of MSCs.

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The aim of this study was to test in vitro the efficacy of TAC, an original G-quadruplex ligand, as a potential radiosensitizing agent for glioblastoma multiforme (GBM). Two human radioresistant telomerase-positive GBM cell lines (SF763 and SF767) were analyzed, with and without TAC treatment, for telomere length, cell proliferation, apoptosis, cell-cycle distribution, gene expression, cytogenetic aberrations, clonogenic survival assay, 53BP1 immunofluorescence staining, and γH2AX phosphorylation. We found that low concentrations of TAC (0.5 and 1 μmol/L) inhibited the proliferation of GBM cells in a concentration-dependent manner after only 1 week of treatment, with minimal effects on cell cycle and apoptosis. TAC treatment had no visible effect on average telomere length but modified expression levels of telomere-related genes (hTERT, TRF1, and TRF2) and induced concentration-dependent DNA damage response and dicentric chromosomes. Survival curves analysis showed that exposure to nontoxic, subapoptotic concentrations of TAC enhanced radiation-induced killing of GBM cells. Analysis of DNA repair after irradiation revealed delayed repair kinetics in GBM cells treated with TAC. Furthermore, the combined treatment (TAC and radiation) significantly increased the frequency of chromosomal aberrations as compared with radiation alone. These findings provide the first evidence that exposure to a G4 ligand radiosensitizes human glioblastoma cells and suggest the prospect of future therapeutic applications. Mol Cancer Ther; 10(10); 1784–95. ©2011 AACR.

Purpose: Genetic instability plays an important role in ovarian carcinogenesis. We investigated the level of telomere shortening and genomic instability in early and preinvasive stages of ovarian cancer, serous tubal intraepithelial carcinoma (STIC), and tubo-ovarian dysplasia (TOD).Experimental Design: Fifty-one TOD from prophylactic salpingo-oophorectomies with BRCA1 or 2 mutation, 12 STICs, 53 tubo-ovarian high-grade serous carcinoma, and 36 noncancerous controls were laser capture microdissected from formalin-fixed, paraffin-embedded sections, analyzed by comparative genomic hybridization (array CGH) and for telomere length (using quantitative real-time PCR based on the Cawthon's method). TOD and STICs were defined by morphologic scores and immunohistochemical expressions of p53, Ki67, and γH2AX.Results: TOD showed marked telomere shortening compared with noncancerous controls (P < 10−7). STICs had even shorter telomeres than TOD (P = 0.0008). Ovarian carcinoma had shorter telomeres than controls but longer than STICs and dysplasia. In TOD, telomeres were significantly shorter in those with BRCA1 mutation than in those with BRCA2 mutation (P = 0.005). In addition, γH2AX expression in TOD and STIC groups with short telomeres was significantly increased (P < 10−7). In dysplastic epithelium, we found subtle genomic alterations, in contrast to more important genomic imbalances in STICs. The total number of genetic alterations was the highest in ovarian cancers.Conclusions: These findings suggest that genetic instability occurs in early stages of ovarian tumorigenesis. STICs and noninvasive dysplasia are likely an important step in early serous ovarian neoplasia. Clin Cancer Res; 19(11); 2873–82. ©2013 AACR.

Supplementary Tables 1-3 - PDF file 74K, aCGH data; minimal regions of interest in tubo-ovarian dysplasia and in STIC; Table S1 : Minimal regions of interest in ovarian dyaplasia (n=5); Table S2 : Minimal regions of interest in the common losses and gains in tubal dysplasia (n=9) 2; Table S3 : Minimal regions of interest in the common losses and gains in STIC (n=12)

International audience; Study question:What are the optimal vitrification method (semi-automated vs. manual) and oocyte stage for in vitro-matured oocyte cryopreservation using meiosis kinetics and chromosome segregation as readouts?Summary answer:The semi-automated vitrification method does not impact oocyte nuclear maturation quality compared with the manual method. Immature oocyte cryopreservation should be performed after IVM.What is known already ?Fertility preservation using oocyte vitrification should be performed before oncological treatments. The reference protocol consists in collecting mature oocytes after ovarian stimulation. Nevertheless, ovarian stimulation sometimes yields immature oocytes or cannot be performed (e.g., emergency oncological treatment). An IVM step is therefore required but it is not clearly demonstrated whether IVM should be performed before or after vitrification. Oocyte vitrification is usually performed with manual methods. A semi-automated vitrification device (Gavi®, Genea Biomedx) showing high performances for embryo was recently released. To our knowledge, no study has analysed its efficiency on oocyte vitrification. Study design, size, duration:200 immature oocytes collected from ICSI cycles from January 2020 will be used. Oocytes will be divided in five groups (40 oocytes/group): freshly matured oocytes (group 1 control), oocytes vitrified after IVM by a manual technique (group 2a) or by Gavi® (group 2b) and oocytes vitrified prior IVM (groups 3a and 3b). We assess oocyte nuclear maturation quality by evaluating IVM kinetics by time-lapse (Geri®) and the accuracy of homologous chromosomes segregation by CGH array. Participants/materials, setting, methods:Since January 2020, 124 out of 200 immature oocytes have been included for this study. These oocytes provide from women under 37 years old without ovulatory disorder after signing an informed consent. The kinetics of meiotic resumption (germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) timings), is determined by time-lapse technology (Geri®, Genea Biomedx). The accuracy of the homologous chromosome segregation during the first meiotic division will be assessed by CGH-Array. Main results and the role of chance:The clinico-biological characteristics (age, BMI, smoking and total FSH dose) are comparable between the five groups (p>0.05). No significant difference in post-thawing oocyte survival rate is observed between the two vitrification methods (semi-automated 58% vs. manual 64%). A significant difference in the overall oocyte survival rate is observed according to the stage of vitrification with a significantly higher survival rate if oocytes are vitrified at the mature stage (93% (2a+2b) vs. 61% (3a+3b), p =0.02). The IVM rate is significantly higher if oocytes are matured freshly (86% in group 1 (fresh IVM, n= 7) and 93% in group 2a+2b (IVM before vitrification, n= 27)) compared to post vitrification (71% in group 3a+3b, n= 28), p=0.03. The vitrification technique does not seem to impact IVM rate since it reached 64% (group 3a, n=11) and 76% (group 3b, n=16) (p=0.4). GVBD and PBE timings are not significantly different between the 5 groups, suggesting that neither the oocyte stage of vitrification nor the vitrification technique affects maturation kinetics. Similarly, our preliminary results of polar bodies and oocytes chromosomal profiles assessed by CGH-Array demonstrate a similar rate of aneuploidy (monosomy or trisomy) between the groups. Limitations, reasons for caution:Our preliminary results of CGH array should be confirmed with the analysis of a larger number of IVM oocytes. Wider implications of the findings:Vitrification of immature oocytes should be performed by semi-automated or manual methods after IVM. Tour knowledge, this is the first study comparing the efficiency of both semi-automated and manual vitrification methods on immature and in vitro-matured oocytes.Trial registration number: NCT03680937

Study question What are the optimal vitrification method (semi-automated vs. manual) and oocyte stage for in vitro -matured oocyte cryopreservation using meiosis kinetics and chromosome segregation as readouts? Summary answer The semi-automated vitrification method does not impact oocyte nuclear maturation quality compared with the manual method. Immature oocyte cryopreservation should be performed after IVM. What is known already Fertility preservation using oocyte vitrification should be performed before oncological treatments. The reference protocol consists in collecting mature oocytes after ovarian stimulation. Nevertheless, ovarian stimulation sometimes yields immature oocytes or cannot be performed (e.g., emergency oncological treatment). An IVM step is therefore required but it is not clearly demonstrated whether IVM should be performed before or after vitrification. Oocyte vitrification is usually performed with manual methods. A semi-automated vitrification device (Gavi®, Genea Biomedx) showing high performances for embryo was recently released. To our knowledge, no study has analysed its efficiency on oocyte vitrification. Study design, size, duration 200 immature oocytes collected from ICSI cycles from January 2020 will be used. Oocytes will be divided in five groups (40 oocytes/group): freshly matured oocytes (group 1 control), oocytes vitrified after IVM by a manual technique (group 2a) or by Gavi® (group 2b) and oocytes vitrified prior IVM (groups 3a and 3b). We assess oocyte nuclear maturation quality by evaluating IVM kinetics by time-lapse (Geri®) and the accuracy of homologous chromosomes segregation by CGH array. Participants/materials, setting, methods Since January 2020, 124 out of 200 immature oocytes have been included for this study. These oocytes provide from women under 37 years old without ovulatory disorder after signing an informed consent. The kinetics of meiotic resumption (germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) timings), is determined by time-lapse technology (Geri®, Genea Biomedx). The accuracy of the homologous chromosome segregation during the first meiotic division will be assessed by CGH-Array. Main results and the role of chance The clinico-biological characteristics (age, BMI, smoking and total FSH dose) are comparable between the five groups (p > 0.05). No significant difference in post-thawing oocyte survival rate is observed between the two vitrification methods (semi-automated 58% vs. manual 64%). A significant difference in the overall oocyte survival rate is observed according to the stage of vitrification with a significantly higher survival rate if oocytes are vitrified at the mature stage (93% (2a+2b) vs. 61% (3a+3b), p = 0.02). The IVM rate is significantly higher if oocytes are matured freshly (86% in group 1 (fresh IVM, n = 7) and 93% in group 2a+2b (IVM before vitrification, n = 27)) compared to post vitrification (71% in group 3a+3b, n = 28), p = 0.03. The vitrification technique does not seem to impact IVM rate since it reached 64% (group 3a, n = 11) and 76% (group 3b, n = 16) (p = 0.4). GVBD and PBE timings are not significantly different between the 5 groups, suggesting that neither the oocyte stage of vitrification nor the vitrification technique affects maturation kinetics. Similarly, our preliminary results of polar bodies and oocytes chromosomal profiles assessed by CGH-Array demonstrate a similar rate of aneuploidy (monosomy or trisomy) between the groups. Limitations, reasons for caution Our preliminary results of CGH array should be confirmed with the analysis of a larger number of IVM oocytes. Wider implications of the findings Vitrification of immature oocytes should be performed by semi-automated or manual methods after IVM. Tour knowledge, this is the first study comparing the efficiency of both semi-automated and manual vitrification methods on immature and in vitro-matured oocytes. Trial registration number NCT03680937

BACKGROUND AND AIMS Genetic etiologies are estimated to affect ∼10% of adults with advanced CKD. However, significant population disparities in genetic kidney disease exist and population-based screening are lacking especially among minority groups. The Druze population is a middle-eastern minority with high rates of end-stage renal disease (ESRD) and consanguinity, which suggests high rates of population-specific monogenic CKD etiologies. We therefore hypothesized that by exome sequencing we will identify a unique distribution of monogenic ESRD causes as compared with prior studies and that important ramifications for clinical practice may ensue. METHOD We initiated a national multicenter study of all Israeli dialysis units in order to provide comprehensive evidence for ESRD's genetic basis (Israeli ESRD Genetic Consortium Cohort). Specifically, during 2020, we recruited 97% (n = 94) of Israeli Druze individuals on dialysis from 12 different hospitals and community-based units. We conducted exome sequencing and diagnostic analysis for all patients. We assessed the diagnostic yield of genetic analysis and its relation to baseline clinical phenotypes. RESULTS Overall, the cohort consists of 94 individuals from 91 different families with first-degree consanguinity rate of 28%. Participants mean age was 62 years (ranging from 18 to 88 years). Most common primary clinical diagnoses were diabetic kidney disease, nephropathy of unknown origin, glomerular or cystic kidney disease, together encompassing 90% of all cases. None had previous molecular diagnosis. Using exome sequencing, we identified a genetic etiology in 27 out of 94 (28.7%) participants. We identified WDR19 (NPHP13) homozygous mutation c.878G > A (p.C293Y) as the most common genetic diagnosis (5.3%). Follow-up clinical characterization revealed that all affected individuals exhibited non-syndromic adulthood-onset ESRD, supporting a profound mutation-dependent phenotypic heterogeneity and weak pre-exome phenotypic specificity. Other prevalent genetic diagnoses included type IV collagen, PKD1, PKD2 and UMOD mutations as well as monogenic forms of diabetes and hyperlipidemia. Molecular diagnosis corresponded to the pre-exome clinical diagnosis in only one-third of the participants. CONCLUSION Exome sequencing in Druze with ESRD yields a genetic diagnosis in just