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O-62 Monday, <strong>October</strong> 19, <strong>2015</strong> 11:30 AM<br />
SUCCESSFUL SURVIVAL IN CULTURE OF SPINAL CORD (SC)<br />
DORSAL ROOT GANGLIA (DRG) NEURONS AND GLIA<br />
CELLS AFTER AUTOMATIC VITRIFICATION. A. Arav, a<br />
A. Shahar, b O. Ziv-Polat, b Y. Natan, c P. Patrizio. d a IVF Research lab, FertileSafe<br />
Ltd., Nes Ziona, Israel; b NVR, Nes Ziona, Israel; c FertileSafe Ltd.,<br />
Nes-Ziona, Israel; d Yale Fertility Center & Fertility Preservati, New Haven,<br />
CT.<br />
OBJECTIVE: To assess the survival and re-growth of neuronal cells after<br />
cryopreservation with a new automatic vitrification system.<br />
DESIGN: Experimental research with SC and DRG isolated from rat fetuses.<br />
MATERIALS AND METHODS: Stationary organotypic DRG and SC<br />
cultures were prepared from rat fetuses (gestational day 15, Lewis inbred,<br />
Harlan, Israel). mmediately after dissection, the DRG and SC were cut<br />
into small slices (400 micron) and seeded in 12 well culture plates containing<br />
1 mL of NVR-Gel. Cultures were enriched once in gel with<br />
GDNF conjugated iron oxide nanoparticles (10 ng/mL) and subsequently<br />
with nutrient medium at each consecutive feeding. Monitoring of the<br />
DRG-SC growth pattern was done by daily phase contrast microscopic<br />
observations 24 hours after setting the cultures onward and by IF staining.<br />
The various neuronal samples (SC, DRG, glia cells) were cryopreserved<br />
with a new device for automatic vitrification (SarahÒ, Fertilesafe, IL),<br />
consisting of a special capsule containing EM gold grids attached to<br />
0.25ml straws (IMV,France). The straws loaded with neuronal tissue<br />
were placed into the mixing chamber of the device attached to a syringe<br />
pump dispensing timed and gradual increasing concentrations of equilibrium<br />
solution for 12-25 min. and vitrification solutions for 3-5 min.,<br />
before plunginginto liquid nitrogen slush (VitMaster, IMT IL). After 1<br />
week the samples were rewarmed according to the Origio’s kit instructions.<br />
To assess survival, the cells recovered were fixed (4% paraformaldehyde),<br />
permeabilized with 0.1% Triton X-100 in PBS and then<br />
immunoblocked with a 1% BSA in PBS for 1 h at RT prior to double<br />
staining with mouse anti S-100 antibodies (1:80, Acris Antibodies, glial<br />
cell marker) and rabbit anti neurofilament antibodies (NF, Novus Biologicals,<br />
1:500, neuronal cell marker). The primary antibodies were diluted<br />
in 0.1% BSA and 0.05% Tween-20 in PBS and incubated with the specimens<br />
overnight at 4 C. After rinsing, the DRG specimens were incubated<br />
for 1 h at RT with fluorescent secondary antibodies.<br />
RESULTS: Organotypic cultures of SC, DRG neurons and glia cells<br />
demonstrated a remarkable survival as well as nerve fiber regeneration after<br />
cryopreservation/rewarming. The SC neurons maintained their multipolar<br />
shape with re-growth of dendrites and axons. The round shaped DRG neurons<br />
exhibited euchromatic nuclei with prominent nucleoli and an active regeneration<br />
of nerve processes.<br />
CONCLUSIONS: This is the first report on successful vitrification of SC<br />
cells, DRG neurons and glia cells using an automatic freezing device. The<br />
remarkable resumption of growth for neuronal cells will greatly expand<br />
regenerative research opportunities.<br />
O-63 Monday, <strong>October</strong> 19, <strong>2015</strong> 11:45 AM<br />
THE IN VITRO DEVELOPMENT OF HUMAN ZYGOTE<br />
RECONSTRUCTED BY PRONUCLEAR TRANSFER. H. Liu, a<br />
Z. Lu, a A. Chavez-Badiola, b P. Colls, c S. Munne, c J. Zhang. a a New<br />
Hope Fertility Center, New York, NY; b New Hope Fertility Center Mexico,<br />
Guadalajara, Jalisco, Mexico; c Reprogenetics, Livingston, NJ.<br />
OBJECTIVE: Nuclear transfer techniques, including metaphase II<br />
spindle transfer and pronuclear transfer (PNT), have been proposed as<br />
potential therapeutic measures to prevent maternal mitochondrial (mt)<br />
DNA disease transmission and to treat recurrent failure of implantation<br />
due to ooplasmic dysfunction. Although PNT has been widely tested on<br />
several animal models resulting in normal offsprings, the use of this<br />
technique in humans is still limited due to safety and efficacy concerns.<br />
This study aimed to examine the feasibility of PNT and its outcome<br />
on human chromosome complements and pre-implantation blastocyst<br />
development.<br />
DESIGN: Two pronuclei (2PN) were exchanged between two zygotes<br />
via PNT; the reconstructed zygotes were cultured to blastocysts followed<br />
by aneuploidy testing using array comparative genomic hybridization<br />
(aCGH).<br />
MATERIALS AND METHODS: Oocytes and sperm from gamete donors<br />
aged between <strong>21</strong> and 28 were collected and frozen for these experiments.<br />
Donor oocytes and sperm were then thawed and cultured in vitro<br />
for 3 hours before intracytoplasmic sperm injection (ICSI). Only zygotes<br />
with 2PN at 15 hours after ICSI were used in this study and were subjected<br />
to PNT via electric pulse to initiate membrane fusion between<br />
the isolated karyoplasm and ooplasm. Reconstructed zygotes were<br />
cultured in vitro until the blastocyst stage. The blastocysts were biopsied<br />
by extracting 3-5 cells of the trophectoderm followed by aCGH testing<br />
for aneuploidy. Blastocysts formed from fertilized donor oocytes and<br />
sperm that were not subjected to PNT were used as controls (non-<br />
PNT). Data were analyzed using chi-square test.<br />
RESULTS: Thirty-two donor oocytes were thawed with 93.8% survival<br />
rate, and 80% of the survived oocytes fertilized normally after ICSI with<br />
thawed donor sperm forming a total of 24 zygotes (2PN). The zygotes<br />
were then subjected to PNT, 37.5% of which developed to the blastocyst<br />
stage compared to 53.7% blastocyst formation rate in the non-PNT group<br />
(p>0.05). In addition, 7 out of 9 (77.8%) PNT blastocysts showed euploid<br />
chromosomes following aCGH compared to 55% (n¼158) euploidy rate in<br />
the non-PNT group (p¼0.6).<br />
CONCLUSIONS: PNT is applicable in human zygotes without imposing<br />
adverse impact onto the pre-implantation embryonic development and its<br />
chromosomal content.<br />
O-64 Monday, <strong>October</strong> 19, <strong>2015</strong> 12:00 PM<br />
IDENTIFICATION OF EMBRYO MARKERS PREDICTING<br />
BLASTOCYST FORMATION BEFORE 1ST CLEAVAGE. Y. Kida,<br />
N. Fukunaga, H. Kitasaka, T. Yoshimura, K. Nakayama, H. Ohno,<br />
M. Takeuchi, M. Shimomura, S. Kounogi, Y. Asada. Asada Ladies Clinic<br />
Medical Corporation, Nagoya, Aichi, Japan.<br />
OBJECTIVE: To identify potential embryo markers predicting blastocyst<br />
formation before 1st cleavage using EmbryoScopeÒ.<br />
DESIGN: Prospective cohort study.<br />
MATERIALS AND METHODS: We examined 288 embryos resulting<br />
from normal fertilization in 25 recipient patients undergoing ICSI between<br />
March and July, 2013. The embryos were placed and cultured in the Embryo-<br />
ScopeÒ in droplets of 30ml of Continuous Single Culture Medium (Irvine<br />
Scientic:USA) till day 7. We analyzed the time of PB2 extrusion (tPB2),<br />
appearance (tPNa) and fade (tPNf) of pronuclei. For tPNa, the time of<br />
maternal pronuclei appearance was used. The following intervals were calculated:<br />
PB2-PNa, PNa-PNf. We used Gardner’s classification to evaluate blastocysts.<br />
Blastocysts evaluated more than 3BB were considered as goodquality<br />
ones. T-test was used for comparison of mean timings and Chisquared<br />
test for comparison of blastocyst rates. P-values
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