title: Enzymatic characterization of zona pellucida hardening in human eggs and embryos.
abstract: OBJECTIVE
  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 alpha-chymotrypsin treatment after 0, 24, 48, 72, 96, 120 h (experiment 1) and 48 h, 72 h, and 1 week (experiment 2) of in vitro culture 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 vitro from 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 variation (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 microns; mean 14.6 +/- 0.2 microns) 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 vitro culture, similar to levels achieved postfertilization. The data do not support, however, the concept that additional, abnormal hardening of the ZP occurs during extended culturing.
authors.affiliation: Department of Obstetrics and Gynecology 92668, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Schiewe
authors.forename: E
authors.initials: E
authors.lastname: Araujo
authors.forename: R H
authors.initials: RH
authors.lastname: Asch
authors.forename: J P
authors.initials: JP
authors.lastname: Balmaceda
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000494:pharmacology*; D002970:Cleavage Stage, Ovum / Q000187:drug effects*; D004527:Egg Proteins / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008875:Middle Aged; D009865:Oocytes / Q000187:drug effects*; D009924:Organ Culture Techniques; D011956:Receptors, Cell Surface; D013997:Time Factors; D015044:Zona Pellucida / Q000187:drug effects*; D000072216:Zona Pellucida Glycoproteins; D015053:Zygote / Q000187:drug effects*
chemical_list: D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; D002918:Chymotrypsin