title: Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse.
abstract: OBJECTIVE
  Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching in vitro?
  
  
  CONCLUSIONS
  No, unlike laser zona opening, zona thinning does not facilitate embryo hatching.
  
  
  BACKGROUND
  Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results.
  
  
  METHODS
  Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy.
  
  
  METHODS
  Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (n = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 μm and width of the thinned area was 35 μm. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student t-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons.
  
  
  RESULTS
  The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, P < 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona-thinned area. Hatching in about one-third of the zona-intact embryos began with breaches at multiple sites by small groups of cells. Likewise, 53.6% of zona-thinned embryos had multiple breaches, always involving an area outside the thinned zone. Zona opening decreased multiple breaching and led to blastocyst escape an average of 14 h earlier than zona-thinned embryos and 5.5 h before control embryos (P = 0.0003).
  
  
  CONCLUSIONS
  The experiments presented here were limited to in vitro experiments performed in the mouse. Whether human embryos would behave the same way under similar circumstances is unknown. We postulate that zona thinning is not beneficial in human embryos.
  
  
  CONCLUSIONS
  The experiments demonstrate that zona thinning is not equivalent to zona opening for assisted hatching. The study provides reason for systematic reviews of assisted hatching trials to take the method of assisted hatching into consideration and not combine the results of zona thinning and zona opening procedures.
  
  
  BACKGROUND
  Institutional funds were used for the study. No competing interests are declared.
authors.affiliation: Tyho-Galileo Research Laboratories, ART Institute of Washington, 3 Regent Street, Suite 301, Livingston, NJ 07039, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Schimmel
authors.affiliation: Tyho-Galileo Research Laboratories, ART Institute of Washington, 3 Regent Street, Suite 301, Livingston, NJ 07039, USA jc@embryos.net.
authors.forename: Jacques
authors.initials: J
authors.lastname: Cohen
authors.affiliation: Independent Consultant, Geneva, Switzerland.
authors.forename: Helen
authors.initials: H
authors.lastname: Saunders
authors.affiliation: Center for Human Reproduction, North Shore University Hospital, 300 Community Drive, Manhasset, NY 11030, USA.
authors.forename: Mina
authors.initials: M
authors.lastname: Alikani
mesh_terms: D000818:Animals; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000648:ultrastructure*; D047108:Embryonic Development; D007834:Lasers; D051379:Mice; D008846:Micromanipulation; D027724:Reproductive Techniques, Assisted; D059008:Time-Lapse Imaging; D015044:Zona Pellucida / Q000502:physiology* / Q000648:ultrastructure
keywords: alternate hatching; assisted hatching; multiple breaching; zona pellucida opening