Enzymatic characterization of zona pellucida hardening in human eggs and embryos

Purpose To characterize possible hardening of the human zona pellucida (ZP) and evaluate the effect of culture duration, patient age, and ZP thickness, ZP of unfertilized eggs (experiment 1, n =367; experiment 2, n =174) and abnormal embryos (experiment 1, n=52) were randomly designated for -chymotrypsin treatment after 0, 24, 48, 72, 96, 120 h (experiment 1) and 48 h, 72 h, and 1 week (experiment 2) of in vitroculture in HTF medium supplemented with 0.5% human serum albumin. Mean ZP thickness was predetermined in experiment 2.Methods The dispersion of the ZP glycoproteins was assessed, and the duration of time for complete digestion was recorded as an index of ZP hardness.Results In experiment 1, enzyme digestion duration increased (P<0.05) in the first 24 h in vitrofrom 18.0±2.0 to 34.6±2.5 min, and tended to decrease over the next 4 days in culture (25.2±1.3, 29.4±0.9, 27.3±0.6, 26.6+1.1, and 20.7±1.5 min on Day 2– 6 ZP, respectively). Zona hardening of fertilized eggs was revealed by a longer (P<0.01) digestion time (32.2±1.8 vs 25.8±0.6 min).Conclusions There were significant patient-to-patient variations (16.4±0.7 to 39.6±2.2 min); however, age was not correlated to enzyme digestion duration. In experiment 2 we determined that ZP thickness (range 8.4–21.6 m; mean 14.6±0.2 m) was not correlated (r=0.09) to the digestion interval (mean 24.3 ± 0.8 min). Based on our enzymatic ZP digestion measurements, it is apparent that spontaneous zona hardening does occur within 24 h of in vitroculture, similar to levels achieved postfertilization. The data do not support, however, the concept that additional, abnormal hardening of the ZP occurs during extended culturing.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.