INTRODUCTION

Cytoplasm fragments can be removed, by delicate suction, after a hole is opened in the ZP. There is no evidence that fragment removal interferes with subsequent pre-embryo development, provided that it is performe in na adequate way; much on the contrary, this procedure can only benefit the pre-embryos that have fragmentation. Certain fragment types are easily removable, particularly those ones free in the perivitellinean space. On the other hand, fragments attached to the blastomeres (which haven’t come loose from their superficies) must be left intact.

During a secure and well succeeded fragment removal we must always keep refocusing fragment and micropipette. Controlled and careful suction is also important during embryo manipulation, speciallly when the micropipette is inside the ZP and sucton is being used to remove the fragment from this space. The most secure method is to remove one fragment at a time and aspire it into the micropipette only at the moment when the pipette is outside the ZP. Fragment removal is a technique that demands time and patience, however, it is not necessary to remove all of them, it is enough if the final result is 5 –10 % fragmentation.

Pre-embryos must be whirled with the holding micropipette to facilitate fragment visualization and remotion. This permits the complete remotion in each area. If fragments are scattered inside the ZP it will be necessary to rotate and reposition the pre-embryo every moment.

Fragments that are free in the perivitellinean space and easy to remove are called FREE, as opposed to those ones with a massive aspect in a determinate area and/or tightly attached to one another and to the blastomeres, which are called MASSIVE (TUFFS). Individual fragments scattered inside the ZP are called DISPERSE, and hard fragments, difficult to aspire and remove are called RIGID.

In rare occasions (< 1% of manipuled pre-embryos) blastomeres can be damaged during fragment removal. When this occurs, the damaged blastomere must be removed. Reasonable pregnancy rates after blastomere biopsy suggest that the removal of a single blastomere won’t affect the subsequent embryo development.

During AHA the external superficies of ZP must be cleaned (remotion of corona cells, granulosa and sperm). This is desirable because those cells go through apoptosis after prolonged in vitro exposition to culture media. A clean embryo has a better morphological appearance after fragment removal.

AHA ON DAY 2

In the special cases when all embryos posses excessive fragmentation (>20%) on day 2 (46-48 hours after insemination), AHA can be done in a precocious time to optimize embryo development potential. The removal of degenerating fragments that may be taking space permits a better growth ambient and improves cell to cell contact. However, removing ragments on day 2 may be more dificult because of a bigger amount of macico fragments and higher degree of adhesion between fragments and blastomeres. Moreover, the simple damage of a single blastomere, among the few available, may impede subsequent embryo development. Nevertheless, despite the dificult and stressful procedure, it has been proven to be beneficial to the pre-embryo development, compared to control pre-embryos not submitted to AHA on the second day.

AHA IN FROZEN/THAWED PRE-EMBRYOS

During freeze and thaw procedure, one or more blastomeres may degenerate and die. The resulting degenerate naterial must be removed after AHA. This will alow tranferring only embryos clean from the material which could hinder their development. Dead or degenerated blastomeres must be removed soon after thawing.

The ZP changes its consistency after prolonged exposition to low temperatures; it is possible that this decurent increase in rigidity impede the natural process of hatching. AHA is indicated, in this case, for those special circumstances. Special care is reccommended in such cases (removal of degenerating blastomeres), due to diminished elasticity of ZP and its tendency to shrink, as well as membrane fragility of non degenerated blastomeres. If breaches in the ZP are observed after thawing, effort must be made to remove degenerated material through the already existing openings, since the presence of multiple openings may impede the natural hatching process. Recent studies show higher implantation rates in thawed embryos submitted to AHA, compared to the ones which have been not.

BLASTOCYST AHA

AHA can be performed in the blastocyst stage. Some expanded blastocysts are not capable to enter hatching naturally, as observed in vitro. AHA is able to recover these blastocysts and prevent them to shrink. However, using Tyrode’s Acid solution (TA) is not reccommended due to thining of the ZP and lack of perivitellinean space. The use of a technique like PZD or other mechanical treatments is preferrable at this developmental stage.

ADVANCE