Mechanical reduction of the blastocoelic cavity before slow cooling cryopreservation does not improve blastocyst survival rates

Abstract

Objective: Mechanical reduction of the blastocoelic cavity has been recently reported by Vanderzwalmen and co-workers (Fertil Steril O-125, S48; Vol. 74:3, Suppl. 1, 2000) to increase blastocyst viability after vitrification. The purpose of our study was to evaluate the effectiveness of this novel technique in our ART laboratory’s slow cooling protocol for blastocyst cryopreservation. Design: Mouse blastocysts (B6C3F1/B6D2F1) with or without mechanical removal of the blastocoelic cavity’s fluid were cryopreserved using a two-step slow cryopreservation protocol, followed by a rapid thawing and a four-step dilution at room temperature. Blastocysts were maintained in culture for 24 hours to assess viability and further development. Materials/Methods: One hundred forty-six mouse blastocysts (n=3) were randomly allocated into two groups. Blastocysts in group A (n=72) were transferred into micro-droplets containing freeze-thaw diluent (modified human tubal fluid with hepes and supplemented with 20 mg/ml human serum albumin) for mechanical reduction of the blastocoelic cavity. For the procedure, each blastocyst was placed with the inner cell mass at either 12 or 6 o’clock and firmly secured with a holding pipet. A micro-needle positioned at 3 o’clock, was slowly inserted through the cellular junction of the trophectoderm into the blastocoele cavity until complete blastocyst compaction was observed. Control blastocysts in group B (n=74) were incubated in freeze-thaw diluent for 15 min. before cryopreservation. The freezing solutions for both groups of embryos were 5% glycerol (10 min.), followed by 9% glycerol and 0.2 M sucrose (10min.). Blastocysts were transferred into cryovials and placed into the freezing machine at 20 °C, then cooled at 2 °C/min. to -6 °C, seeded and held for 10 min., followed by cooling at 0.3 °C/ min. to -36 °C and plunged into liquid nitrogen for storage. Blastocyst thawing was accomplished by placing the cryovials in a 35 °C water bath, then transferring the blastocysts into 6% glycerol with 0.4 M sucrose, 3% glycerol with 0.2 M sucrose, 0.1 M sucrose, and freeze-thaw diluent at five minutes intervals. Embryos were rinsed with G2.2 medium and incubated overnight in micro-droplets overlayed with mineral oil. Data were analyzed using ANOVA and chi-square. Results: Post-thaw survival was 73.6% for blastocysts in group A and 68.91% in group B. At 24 hours, complete blastocyt expansion occurred in 86.7% of the blastocysts with mechanical shrinkage and in 70.5% in the control group. Blastocyst hatching was observed in 52.8% of the surviving embryos in protocol A and 47% in protocol B. No significant statistical differences between group A and B were observed for any of the parameters evaluated. Conclusions: The observations reported here suggest that mechanical reduction of the blastocoelic cavity before slow cooling cryopreservation neither appears to increase blastocyst viability nor has a deleterious effect. Supported by: Center for Reproductive Health.