Revisiting embryo assisted hatching approaches: a systematic review of the current protocols

Abstract

Zona pellucida (ZP) manipulation, termed "assisted hatching" (AH), has been introduced in order to favor embryo hatching and ultimately improve assisted reproductive technology success but with poor proofs of safety and biological plausibility. We herein provide a systematic review of clinical outcomes following the application of different methods of ZP manipulation on fresh or frozen/thawed embryos at different developmental stages in different groups of patients. Out of the 69 papers that compared the clinical outcomes deriving from hatched versus non-hatched embryos, only 11 considered blastocysts while the rest referred to cleavage stage embryos. The ZP thinning of fresh embryos either by chemical or laser approach was shown to provide very limited benefit in terms of clinical outcomes. Better results were observed with procedures implying a higher degree of zona manipulation, including zona removal. Studies comparing the mechanical or chemical procedures to those laser-mediated consistently reported a superiority of the latter ones over the former. Literature is consistent for a benefit of ZP breaching in thawed blastocysts. This review provides the current knowledge on the AH procedure in order to improve its efficacy in the appropriate context. Embryologists might benefit from the approaches presented herein in order to improve Assisted Reproduction Technologies (ART) outcomes.

Keywords: Assisted hatching; Embryo culture; Hatching; Implantation failure; Zona hardening; Zona pellucida.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Flow diagram of the search strategy, screening, eligibility, and inclusion criteria

Fig. 2

Trapping events. Blastocysts were laser-hatched to favor the biopsy. a The embryo is hatching from two openings. b, c The embryo splits in two parts with the ICM trapped and degenerated into the ZP

Fig. 3

Procedure commonly used for embryo chemical thinning: [1] the embryo is held by a holding pipette and approached by an AH needle filled by acidified/enzymatic solution; [2] the solution is released gradually in vertical fashion; [3] the solution release is stopped immediately once desired thinning is achieved

Fig. 4

Procedure commonly used for embryo chemical breaching: [1] the embryo is held by a holding pipette and approached by an AH needle filled by acidified solution; [2] the solution is released gradually and from a distance to assure no harm occur to embryos; [3] the solution release is stopped immediately once a hole is created

Fig. 5

Procedure commonly used for embryo PZD: [1] the embryo is rotated with the microneedle until the area with the largest perivitelline space is visible at the 12 o’clock position; [2] ZP is pierced by a needle at the 1 to 2 o’clock position; [3] the embryo is released from the holding pipette and rubbed against it until the embryo is released

Fig. 6

Procedure commonly used for embryo 3D-PZD: [1] the embryo is rotated with the microneedle until the area with the largest perivitelline space is visible at the 12 o’clock position; [2] the ZP is pierced by a needle at the 1 to 2 o’clock position; [3] the embryo is released from the holding pipette and rubbed against it until the embryo is released; [4] the embryo is held again and rotated vertically until the first slit is visible at the 12 o’clock position; [5] steps from 1 to 3 are repeated until the embryo is released from the piercing needle

Fig. 7

Procedure commonly used for embryo LZD: [1] the embryo is held at about the 8 o’clock position and then is pierced with the tip of the hatching needle at about the 5 o’clock position. Pushing the cell membrane aside by using the needle tip allows to obtain space in the perivitelline area; [2] the needle advances until it touches the opposite side of the ZP; [3] once there, the needle is withdrawn a little from the ZP, then the embryo is released from the holding pipette and rotated; [4] the embryo is then held from a new site and the needle is moved forward to cut the ZP. Repeating the step of rotating the embryo and piercing a new site helps obtaining a cut ZP with a controlled size; [5] the curve of the needle is pushed against the bottom of the dish to cut the pierced ZP

Fig. 8

Procedure commonly used for embryo laser HA. The embryo is thinned with a single hole without reaching the inner membrane [a] or is thinned irradiating at one point and continuing until one quarter [b] or half [c] of ZP; the embryo is breached with a single hole completely through the ZP [d], or is breached for one quarter [e] or for half of ZP [f]; the blastocyst is breached with a single hole completely through the ZP [g], or is breaching for one quarter [h] or for half of ZP [i]