P-56: Validation of vitrification for clinical use: Post-warming in vitro maturation and blastocyst outgrowth

Abstract

The effectiveness of vitrification as a method for cryopreservation of oocytes and day 5 blastocysts was studied prior to introduction of vitrification for routine clinical use. Extranumerary oocytes and blastocysts were donated for research with informed consent by infertility patients after conclusion of treatments. Vitrified immature oocytes at Germinal Vesicle (GV) or Metaphase I (MI) stage were warmed, placed into maturation medium for 24-72 hours and observed for progression to Metaphase II (MII) stage. Oocytes that reached MII stage in vitro were activated with calcium ionophore to demonstrate functional integrity. Vitrified MII oocytes were activated as controls. Previously vitrified day 5 blastocysts were warmed and cultured for an additional 8 days in vitro. Blastocyst outgrowth size and βhCG production was analyzed at 48 hour intervals. Oocytes and day 5 blastocysts were vitrified in hemistraws using 10% dimethylsulfoxide (DMSO), 10% ethylene glycol in modified Human Tubal Fluid medium (mHTF) with 10% human serum albumin (HSA) for 2 minutes followed by brief exposure to 20% DMSO, 20% ethylene glycol, Ficoll, 0.65M sucrose in mHTF with 10 % HSA before loading hemistraw and plunging into liquid nitrogen. Vitrified oocytes and blastocysts were warmed in 37°C 0.5M sucrose in mHTF, then immediately placed in 0.25M sucrose (5 min), 0.125M sucrose (5 min) and final wash in mHTF. Warmed oocytes were cultured in maturation medium (G2 medium with HSA, 1 IU/mL FSH, 1IU/mL LH, 9 IU/mL hCG, 1 μg E2). Artificial activation of MII oocytes was performed by exposure to 5μM calcium ionophore A23187 and 10 μg/ml puromycin as previously described by Nakagawa et al., (Fertility and Sterility 76:1, 2001). Blastocysts were cultured in 0.5 ml G2-plus medium with 10% heat inactivated FBS in an organ culture dish for 8 days. At 48 hrs intervals, conditioned medium was stored at -20C until analysis and replaced with 0.5 ml fresh medium. Outgrowth size was measured and photographed at 48 hour intervals. Conditioned medium was analyzed for βhCG levels using a solid-phase two-site chemiluminescent immunometric assay (DPC quantitative βhCG kit LKCG1). Control medium consisted of medium cultured for the same interval without blastocyst exposure. Vitrified immature oocytes were able to undergo in vitro maturation and artificial activation by calcium ionophore upon warming and continued culture. Of 5 vitrified GVs, 4 (80%) survived warming, 75% (3) of these progressed to MII in culture and 100% (3) activated when challenged with calcium ionophore. Activation was demonstrated by the formation of pronuclei. Of 12 vitrified MI stage oocytes, 75% (9) survived warming, and 66% (6) progressed to the MII stage and 50% (3) were activated by calcium ionophore. Vitrified MII oocytes were activated as positive controls. Warmed day 5 blastocysts exhibited attachment and a mean 5-fold increase in outgrowth diameter (221 microns at 48 hrs to 1085 microns at 192 hours). Increasing concentration of βhCG per attached blastocyst (15 ng/ml at 48 hours to 11,942 ng/ml at 192 hours) was also detected in the conditioned medium. Vitrification and warming of oocytes and blastocysts preserved functional integrity of both oocytes and blastocysts as demonstrated by in vitro maturation to MII stage with artificial activation and blastocyst outgrowth with βhCG production.