“In Vitro” Survival of Metaphase II Oocytes (MII) and Blastocysts After Vitrification in a Hemi-Straw (HS) System

Abstract

Objective: Vitrification of MII oocytes and blastocysts in French mini-straws has for several years been considered as an attractive cryopreservation option. In order to increase the efficacy of vitrification, protocols were introduced to increase the cooling and warming rate up to 2 × 104°C/min. Different promising vitrification methods using open pulled straws, electron microscope copper grids, nylon loops system have been developed. But it remains that the storage was less practical when these methods are used. To facilitate the storage, we developed a simple and efficient method of vitrification using a modified straw system, allowing direct contact between the oocytes or embryos and liquid nitrogen. Design: The effectiveness of the Hemi-straw vitrification procedure was studied on 46 MII oocytes and on 16 blastocysts. Materials and Methods: HS used for vitrification consists of a 250 μl French straw cleaved in length. Day 1 unfertilized oocytes (n=27) and oocytes in MII (n=19) that were not inseminated, spermatozoa not being available at the time of oocyte retrieval were included in the vitrification program. Blastocysts (n=16) not suitable for conventional slow freezing protocol were also vitrified with the HS procedure. The vitrification solution consists in a mixture of ethylene glycol (EG)-DMSO. After an equilibration of 2 minutes in EG (7.5%)-DMSO (7.5%), the oocytes or blastocysts were immersed in EG (15%)-DMSO (15%) for 15 seconds and suspended on a thin film on the HS. Before immersion into the liquid nitrogen, the HS was loaded into a larger straw (500μl). For thawing, the straws were immersed into sucrose 0.5M before dilution into medium. After thawing, the rate of intact unfertilized and MII oocytes was recorded. ICSI was performed on intact MII oocytes. Fertilization and cleavage was followed for 3 days in G1 medium (IVF Scandinavian Science). “In vitro” development of blastocyst was assessed in G2 medium for 48 hrs. Results: Tabled 1OocytesN°Intact after thawingICSI2PNCleaved embryosBlasto- cystsExpandedHatchDay 1 un- fertilized2720 (74%)1614 (88%)7 (44%)M II1914 (74%)1412 (63%)10 (52%) Open table in a new tab Conclusions: Vitrification using the HS protocol showed encouraging results in terms of survival after thawing for both oocytes and blastocysts. Furthermore the percentage of fertilization and zygote developing to embryos is high. The HS protocol did not reduce the ability of the blastocysts to reexpand and hatch in culture. This technique in straw is a promising aid in cryopreservation of oocytes and allows, in addition an easier storage as compared to the cryo-vial system. However, it remains that for oocytes careful investigations have to be performed at the level of aneuploidy and cytoskeletal structure and for blastocysts cell counting have to be evaluated according to the time spent into the LN2.