Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush.

BACKGROUND: Vitrification is becoming the method of choice for embryo cryopreservation. Nevertheless, major problems are still associated with this process such as chemical toxicity and osmotic stress as well as risk of liquid nitrogen (LN) contamination. METHODS: An innovative vitrification method that combines LN slush and sealed pulled straws (SPS) was employed to achieve a high cooling rate, enabling a reduction in cryoprotectant concentration. Open pulled straws were sealed at both ends to prevent direct contact with LN. RESULTS: Ultrarapid cooling of murine embryos at 32 200 degrees C/min in SPS with LN slush yielded a higher blastocyst survival rate (54 +/- 3.5%, 106/196) than cooling at 1700 degrees C/min in 0.25 ml straws (10 +/- 2.1%, 21/197) (P < 0.05). Embryos at the 2-cell stage cryopreserved in 75% vitrification solution (VS) (100% VS contains approximately 5 M ethylene glycol, 0.6 M trehalose and 6% w/v bovine serum albumin) in SPS formed blastocysts at a higher rate (79 +/- 3.6%, 99/126) than cryopreservation in 100% VS (31 +/- 6.5%, 16/51), however, this was not significantly different from the fresh control group (88 +/- 4.6%, 43/49). Early stage embryos at the 2 pronuclei- and 4-8-cell stage formed blastocysts at rates of 68 +/- 4.5 and 60 +/- 3.7%, respectively, after vitrification in 87.5% VS. CONCLUSIONS: This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.

Prediction of embryonic developmental competence by time-lapse observation and 'shortest-half' analysis.

Selecting an embryo with the highest probability of achieving a pregnancy is a major challenge. Early-cleavage embryos are considered to be of good quality; however, the exact developmental stage that predicts further development has not been defined. The aim of the study was to characterize cleavage rate and distribution of various stages of mouse preimplantation embryos using a time-lapse system. Mated mice were killed 20 h after human chorionic gonadotrophin administration and putative zygotes were recovered and cultured in an incubator-enclosed time-lapse imaging system. The 'shortest half' analysis was used to establish the period in which at least 50% of the embryonic population cleaved within the shortest time. Analysis indicated that through embryonic development, cleavage timing becomes less uniform and the 'shortest half' becomes longer with intervals of 2, 2.5, 3.5 and 5 h for 2-, 4-, 8-cell embryo and blastocyst stages, respectively. The 'shortest half' for the first cleavage was closely synchronized, with 80% of embryos developing to the blastocyst stage. Moreover, slow-cleaving embryos approaching the 2-cell stage expressed inferior developmental potential in comparison to those cleaving within the 'shortest half'. Thus, embryonic cleavage rate seems to be a biological indicator of developmental potential and may be useful for embryo selection.

New trends in gamete's cryopreservation.

We developed new techniques to improve freezing and vitrification of sperm, oocytes and embryos. Our novel freezing technology is based on 'Multi-Thermal-Gradient' (MTG) freezing that is used for sperm. The freezing apparatus has the ability to control ice crystals propagation by changing thermal gradient or the liquid-ice interface velocity which optimizes ice crystals morphology during freezing of cells and tissue. Using this apparatus we were able to freeze bull, stallion, boar, ram, fowl and human sperm with normal post-thaw motility/pre-freezing motility of 70-100%. The vitrification method includes the cooling of nanoliter sample (the 'Minimum Drop Size' technique) in 'super-cooled' liquid nitrogen (-210 degrees C), which maximized cooling rate to the highest physically possible (24-130000 degrees C/min). Using this method we achieved very high survival of bovine oocytes and embryos. Vitrification of oocytes at the MII stage resulted with cleavage and blastocyst rate of 50 and 20%, respectively. The vitrification of in-vitro production (IVP) of bovine embryos allowed the production of a healthy calf after embryo-transfer carrying the name 'Zegugit' (in Hebrew: made from glass).

Whole sheep ovary cryopreservation and transplantation.

This is the first report of successful cryopreservation and transplantation of an intact ovary in a large animal (sheep). Three of eight transplanted sheep had resumed hormonal cyclicity.

The effect of chilling on membrane lipid phase transition in human oocytes and zygotes.

BACKGROUND: Chilling injury occurs when the cell membrane undergoes a transition from the liquid state to the gel state. Human oocytes and single-cell zygotes are of similar shape and size but the post-thawing survival rate of oocytes is poorer. We set out to investigate the possible difference in membrane lipid phase transition (LPT) temperature between the two cell types. METHODS: The LPT temperature was measured with a Fourier Transform Infrared analyser, which detects the change in the vibration frequency of the CH2 bond stretches of the membrane lipid molecules during temperature change. The LPT temperatures of unfertilized human oocytes, in vitro-matured oocytes, and immature germinal vesicle (GV) stage oocytes were compared with that of abnormally fertilized human zygotes. RESULTS: The LPT temperatures of zygotes and of mature and immature GV oocytes differ significantly from each other (10.0 +/- 1.2, 16.9 +/- 0.9 and 24.4 +/- 1.6 degrees C respectively; P < 0.05). CONCLUSIONS: Zygotes show a higher resistance to chilling injury compared to oocytes at different developmental stages; this might explain the relatively poor survival rates of cryopreserved human oocytes and indicates the necessity to adjust the cryopreservation protocols in order to minimize cryoinjury.