Vitrification solution without sucrose for cryopreservation in mouse blastocysts.

OBJECTIVE: This study was designed to investigate the survival rate of vitrified mouse blastocysts depending on the presence or absence of sucrose in vitrification solution. METHODS: Mouse two-cell embryos were collected and cultured to blastocysts. Two vitrification solutions were prepared. The control solution was composed of 25% glycerol, 25% ethylene glycol, and 0.5 M sucrose (G25E250.5S) containing 2.5 mL glycerol, 2.5 mL ethylene glycol, 2 mL SSS, and 0.855 g sucrose in 5 mL PB1. The experimental solution was composed of 25% glycerol and 25% ethylene glycol (G25E25) and contained 2.5 mL glycerol and 2.5 mL ethylene glycol in 5 mL PB1. Artificial shrinkage was conducted by aspirating the blastocoelic fluid using an ICSI pipette. To examine the effect of sucrose in the vitrification solution on the survival rate of mouse blastocysts, the shrunken-equilibrated blastocysts were rehydrated or vitrified after being exposed to one of the two vitrification solutions. After exposure and the vitrification-thawing process, the re-expansion rate and hatching rate were evaluated after 6 hours of in vitro culture. RESULTS: The re-expansion rate of mouse blastocysts exposed to vitrification solution with and without sucrose were not different in the experimental solution (without sucrose) (98%) and the control solution (with sucrose) (92%) (p>0.05). The hatching rate was higher in the experimental solution (95%) than in the control solution (88%), but did not differ across two treatments (p>0.05). The re-expansion rate of mouse blastocysts vitrified in the control solution was 92% and 94%, respectively (p>0.05), and the hatching rate was higher in the experimental solution (90%) than in the control solution (74%) (p<0.05). CONCLUSION: Sucrose need not be added in vitrification solution for freezing of artificially shrunken mouse blastocysts.

The effect of long zona dissection using ICSI pipettes for mechanical assisted hatching in vitrified-thawed blastocyst transfers.

OBJECTIVE: To investigate the effect of long zona dissection (LZD) compared with partial zona dissection (PZD) using ICSI pipettes for mechanical assisted hatching (AH) in vitrified-thawed blastocyst transfers. DESIGN: Prospective study. SETTING: University IVF clinic. PATIENT(S): A total of 120 women ≦ 38 years old undergone vitrified-thawed blastocyst transfers with LZD or PZD. INTERVENTION(S): The culture of all pronucleate embryos to the blastocyst stage and the selection of blastocysts ≧ grade 3BB (Gardner and Schoolcraft score), followed by vitrified-thawed blastocyst transfers with LZD (n = 60) or with PZD (n = 60) MAIN OUTCOME MEASURE(S): Complete hatching rates, implantation rates, pregnancy rates. RESULT(S): At 5 h after thawing, complete hatching rates of blastocysts were significantly higher in LZD group compared with PZD group, 52.4 % vs. 31.8 % (P = 0.001). Implantation and clinical pregnancy rates were significantly higher in LZD group compared with PZD group, 40.9 % vs. 25.7 % and 63.0 % vs. 40.0 %, respectively (P = 0.010, P = 0.011). CONCLUSION(S): LZD using ICSI pipettes for mechanical AH improves significantly complete hatching, implantation and pregnancy rates in vitrified-thawed blastocyst transfers.

Preliminary clinical outcome of novel strategy for the maximization of cumulative pregnancy rates per retrieval in normal responders.

OBJECTIVE: We devised a novel strategy, a GnRH antagonist protocol with a GnRH agonist trigger followed by frozen-thawed blastocyst transfers with long zona dissection (LZD). The purpose of this study was to investigate the clinical outcomes of this new strategy according to age. METHODS: Ninety women aged less than 35 (group A) and 32 women aged 35 to 39 (group B) underwent the GnRH antagonist protocol with a GnRH agonist trigger in order to obtain many oocytes and prevent early-onset ovarian hyperstimulation syndrome (OHSS). All oocytes were cultured to the blastocyst stage and all blastocysts grade 3BB or better were cryopreserved. Embryo transfers were only performed in freeze-thaw cycles to prevent late-onset OHSS and to overcome embryo-endometrium dyssynchrony. LZD was performed just after thawing to improve hatching and implantation rates. RESULTS: The average numbers of retrieved oocytes and blastocysts grade 3BB or better were 12.8±5.5 and 4.4±2.6 in group A and 10.9±7.4 and 2.5±2.2 in group B, respectively, and OHSS did not occur in any of the women. Implantation rates were 46.7% in group A and 39.3% in group B. Cumulative clinical pregnancy rates per retrieval were 77.8% in group A and 62.5% in group B. Cumulative ongoing pregnancy rates per retrieval were 71.1% in group A and 53.1% in group B. CONCLUSION: GnRH antagonist protocol with GnRH agonist trigger followed by frozen-thawed blastocyst transfers with LZD can generate many blastocysts without OHSS and maximize cumulative pregnancy rates per retrieval. This strategy is more effective in young women aged less than 35 than in women aged 35 to 39.