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

Abstract

Enzymatic treatment of the zona pellucida to either soften or remove totally the zona before blastocyst transfer has resulted in high implantation rates. The zona is usually completely dissolved after 1.5 min exposure with 10 IU pronase at 37 degrees C. Since there may be concerns that pronase treatment for periods of 1.5 min or longer may cause adverse effects on the trophectoderm (TE) and inner cell mass (ICM), the changes to human blastocysts exposed to different time intervals of pronase were investigated. Of 18 blastocysts exposed to pronase for 1.5 min, the zona was completely dissolved and no changes were observed by light microscopy (LM) or transmission electron microscopy (TEM), compared with 11 naturally hatched untreated blastocysts (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 tonofibrils attached to desmosomes were observed. When three other blastocysts 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 cells was extensively vacuolated and many TE cells showed cytoplasmic blebbing towards the blastocoele. However, the epithelium was uninterrupted with intact 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 pronase for 1.5 min prior to extensive degeneration taking place. The results confirm that the optimal time for softening or complete removal of the zona before transfer was around 1.5 min and that enzymatic treatment was a safe, non-invasive procedure to remove the zona of blastocysts. The human embryonic TE is a very hardy, robust epithelium that withstands pronase treatment.

Mitochondria directly influence fertilisation outcome in the pig

“…Both zona pellucida (ZP) and attached sperm were removed by exposing each fertilised or unfertilised oocyte to 10 U/ml pronase in IVP media for 1-1.5 min (Fong et al 2001). ZP-free unfertilised oocytes and intact embryos were washed several times in IVP media and transferred to 0.2 ml PCR tubes (Appleton Wood Laboratory Equipment, Birmingham, UK).…”

Derivation, growth and applications of human embryonic stem cells

“…Added to serum-free medium, LIF increases the blastocyst rate, hatching rate and cell number of mammalian preimplantation embryos (Fry 1992, Dunglison et al 1996 but whether greater numbers of ICM cells in day 8 blastocysts allow more-efficient derivation of hES cells is difficult to say. When plated on feeder cells, hES clumps with 50 -100 hES cells have more chances to attach and proliferate than smaller cell clumps , and for successful derivation of hES cells the ICM has to be placed on feeders as a cell clump not as single cells since ICM cells are held together tightly with numerous junctional complexes (Fong et al 2001). Therefore, we believe that the third step could be beneficial in the derivation of hES cells since it allows further proliferation of ICM cells within the blastocyst and allows the ICM cells a further 2 days to adapt to the culture conditions they will be subject to after isolation.…”

Estimating zona pellucida hardness under microinjection to assess oocyte/embryo quality: Analytical and experimental studies

“…On the other hand, with IVF/ICSI treatment, a high proportion of morphologically normal blastocysts have hatching difficulties [10] due to failure of the embryonic ZP puncture following blastocyst expansion [11]. Several investigators believe that failure to hatch is due mainly to abnormal changes in ZP quality, such as thickening or hardening [12][13][14].…”