Korean J Obstet Gynecol.  1999 Nov;42(11):2426-2433.

Biochemical Assisted Hatching of Embryos in Human IVF-ET Program

• Roh SI
• Lee JE
• Lee DR
• Yoon HS

Abstract

Human assisted reproductive technology programs have been being developed marvelously during this decade. However, implantation rates following embryo replacement remain low, regardless of increased fertilization rates by ICSI. One proposed possibility limiting the successful implantation is an impaired hatching caused by suboptimal culture conditions. As to improve the hatching potential of blastocysts, assisted hatching by an artificial alteration of zona pellucida(ZP) have been done in many laboratories using the various methods. We tried to investigate whether the supplementation of proteases into culture media has any effect on development, zona structure, and/or hatching of mouse embryos. Supplementation of either pronase E(PE) or proteinase K(PK) in culture media did not affect development up to blastocyst but significantly increased hatching rate. And we observed the alteration of ultrastructure and casein binding properties of ZP in mouse embryos. Also we investigated the effects of protease on development of human embryos and pregnancy rates in human ART program. From July 1994 to December 1996, 792 cycles(for study I) and 1095 cycles(for study II) undergoing the IVF-ET program in MizMedi Hospital were randomly selected for BAH. The concentrations of proteases used in this study were 1microgram/ml PE, 0.1microgram/ml PK and 1microgram/mlPE+0.1microgram/ml PK in HTF with 0.5% human serum albumin(HSA), and in vitro fertilized embryos were cultured for 24 hours. We analyzed the efficiency and stability of biochemical assisted hatching(BAH) according to the clinical profiles of patients and fertilization methods. After cultured in HTF with proteases for 24 hours of human embryos, the thinning in zona pellucida of embryos was observed but its development was not disturbed. Also, clinical pregnancy rates were higher in the PE, PK and PE+PK groups than the control group without proteases(36.0%(32/89), 35.3%(36/102), 35.1%(39/111) vs. 25.5%(125/490), p<0.05). The live birth rate in the PE, PK and PE+PK groups were increased than control, and the abortion rate were not different. They were showed a effect and safety of proteases treatment in human embryos. We selected PE as BAH for study II because of slightly better embryonic morphology and pregnancy rate. In patients over 35 years old, clinical pregnancy rates of the BAH group was higher than that of the control group(31.4%(58/185) vs. 22.2%(51/230); p<0.05). And in the cases with few oocytes retrieval, or less than 3 cycles of IVF-ET, clinical pregnancy rates of the BAH group was significantly higher than that of the control group(36.8%(86/234) vs. 27.2%(93/342), p<0.05; 36.8%(148/402) vs. 29.9%(168/562), p<0.05). In BAH groups, the clinical pregnancy rate was similar between conventional IVF and ICSI group. From above results, it is suggested that improved hatching by protease treatment is due to physiological alteration of ZP structure, giving rise to the similar hatching process to that in vivo. We suggest that BAH using protease is a simple, safe and economic technique compared to the other known assisted hatching techniques in human ART program.

MeSH Terms

Abortion, Induced
Adult
Animals
Blastocyst
Caseins
Culture Media
Embryonic Structures*
Female
Fertilization
Herpes Zoster
Humans*
Live Birth
Mice
Oocytes
Peptide Hydrolases
Pregnancy Rate
Pronase
Reproductive Techniques, Assisted
Sperm Injections, Intracytoplasmic
Zona Pellucida
Caseins
Culture Media
Peptide Hydrolases
Pronase