Ultrastructural observations of enzymatically treated human blastocysts: zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties

Enzymatic treatment of the zona pellucida to either soften or remove totall y the zona before blastocyst transfer has resulted in high implantation rat es. The zona is usually completely dissolved after 1.5 min exposure with 10 IU pronase at 37 degreesC. Since there may be concerns that pronase treatm ent for periods of 1.5 min or longer may cause adverse effects on the troph ectoderm (TE) and inner cell mass (ICM), the changes to human blastocysts e xposed to different time intervals of pronase were investigated. Of 18 blas tocysts exposed to pronase for 1.5 min, the zona was completely dissolved a nd no changes were observed by light microscopy (LM) or transmission electr on microscopy (TEM), compared with 11 naturally hatched untreated blastocys ts (controls), In another five blastocysts exposed to pronase for 2 min, no LM changes were observed but subtle TEM changes such as fewer bundles of t onofibrils attached to desmosomes were observed, When three other blastocys ts were exposed to pronase for 5 min, the blastocoele collapsed, and the TE cells started to show blebbing under LM, Under TEM, the cytoplasm of TE ce lls was extensively vacuolated and many TE cells showed cytoplasmic blebbin g towards the blastocoele. However, the epithelium was uninterrupted with i ntact tight junctions and desmosomes. Of a separate group of 44 blastocysts cultured in vitro, 54.5% had hatching difficulties when monitored from day 5 to day 8 and 80% of these could be rescued by removal of the zona with p ronase for 1.5 min prior to extensive degeneration taking place. The result s confirm that the optimal time for softening or complete removal of the zo na before transfer was around 1.5 min and that enzymatic treatment was a sa fe, non-invasive procedure to remove the zona of blastocysts. The human emb ryonic TE is a very hardy, robust epithelium that withstands pronase treatm ent.