SUCCESSFUL PRODUCTION OF NORMAL EUPLOID HUMAN BLASTOCYSTS DERIVED FROM SPERM AFTER PARTIAL FREEZE DRYING, REHYDRATION AND ICSI: TOWARDS DEVELOPING A NOVEL METHOD FOR SAFE STORAGE OF BIOLOGICAL SAMPLES

Current cryopreservation and storage methodologies have many unresolved issues including impact of cryoprotectants (CP) on cell survival, Liquid Nitrogen (LN) tank failures which may jeopardize samples integrity during storage, cumbersome and costly sample shipping. Here we aimed at replacing LN storage with ordinary freezers (-80˚C) after partial sperm freeze/drying (pFD) with a CP-free solution. Rehydrated sperm were evaluated for motility and embryo competence following ICSI. Prospective, experimental basic research. Donor sperm cryovials (n=12) were thawed and motile sperm recovered using the ESep device (FertileSafe Ltd, Israel). The separated motile sperm fractions were washed with a solution (Lyo4) containing 0.25M Trehalose and 0.25M Sorbitol in 10mL sperm wash (SW) and diluted 2:1 with SW. Volumes of 100 μL were pipetted into cooled glass vials (-25˚ C). Five samples were placed in the -80˚C freezer for 1 hour (frozen samples) and the other 7 samples underwent partial freeze drying (pFD). pFD was done with a lyophilizer device (Darya, FertileSafe Ltd, Israel) having preset shelf temperature at -25C˚ and operated at a vacuum of <500 mTorr for 10-20 minutes. Samples were then stored at -80˚C for various time lengths (1 to 8 days). Then pFD samples were rehydrated, assessed for motility and used for ICSI with donor MII oocytes. Thawing of frozen samples was obtained by plunging the vials in a 37˚C water bath (1 min) and sperm suspension diluted with same volume of SW. Rehydration after pFD, was done by adding pre-warmed Sperm Wash solution (volume 1:1) at 37˚C to the sperm pellet. Sperm motility was evaluated (i) at the baseline thawing after ESep preparation of the donor sperm cryovials; (ii) after Lyo4 addition and freezing/thawing and (iii) after pFD after different storage-times. Both immotile and motile sperm after freezing/thawing and pFD were selected for ICSI of donor MII oocytes. Embryo aneuploidy screening was conducted via Non-Invasive Chromosome Screening (NICS) with DNA obtained from embryo culture media utilizing previously validated NICS platform. ESep processed sperm cryopreserved at -80⁰C in CP-free Lyo4 maintained had high post thaw motility (94%) at 1 hour, supporting the choice of Lyo4 as the optimal SW protocol for sperm pFD. Preliminary work (data not shown) had demonstrated that pFD-sperm without Lyo4 had no survival. Upon thawing and rehydration pFD-sperm had the following motility: 69% at 24 hrs; 43% at 7 days and 6% at 8 days. After ICSI with immotile sperm (n=6), there were 0 fertilized oocytes and 0 blastocysts; after ICSI with motile PFD-sperm (n=13) there were 8 correctly fertilized oocytes, 6 blastocysts, 3 euploid (46XX). This experiment demonstrated that sperm selected by ESep after partial freeze drying with CP-free solution maintains motility during storage at -80⁰C for various lengths of time. To our knowledge this is the first report that using rehydrated motile sperm for ICSI produces human euploid blastocysts.