Relevance of Assisted Hatching with Blastocyst Stage Transfer


When a blastocyst expands with the growth of the blastocoel, it stretches and thins the zona pellucida (ZP), ultimately rupturing the ZP allowing the embryo to hatch free ready for implantation.

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When a blastocyst expands with the growth of the blastocoel, it stretches and thins the zona pellucida (ZP), ultimately rupturing the ZP allowing the embryo to hatch free ready for implantation. In the event this does not occur, then an otherwise potentially viable embryo may not implant. Assisted hatching (AH) has been developed to overcome this, but has been applied mostly to day-2/3 embryos (Cohen et al, 1992). More recently it has been reported that total zona removal with pronase can be successfully undertaken with human blastocysts (Fong et al, 1998), to enable “naked” fully-hatched blastocysts to be transferred to the uterus ready for immediate implantation. By taking a less aggressive approach with acidified Tyrode’s medium, it is equally possible to drill a large hole in the ZP of a blastocyst, without the risks associated with global exposure of the embryo to pronase. Initial retrospective comparison of AH applied in this manner to the “slower” day-6 blastocysts suggests a distinct benefit in terms of implantation when compared to non-AH control blastocysts.


Extended culture of human embryos to the blastocyst stage enables increased selection potential of embryos for transfer. This achieves improved pregnancy rates, higher implantation rates per embryo transferred allowing fewer embryos to be replaced into the uterus, thus decreasing the risk of multiple implantation (Gardner et al, 1998). Additionally, the feasibility of growth to the blastocyst stage itself can act as a diagnostic tool, and as a potential passive screen for certain aneuploidies (Jones & Trounson, 1999). In vitro culture may continue for 5 or 6 days depending on the rate of development of the embryos to the blastocyst stage. This appears very patient specific, and is probably related directly to the viability of individual embryos. AH has been shown to accelerate implantation (Liu et al, 1993), probably through earlier release of the embryo from its ZP. Hence, delayed development of day-6 blastocysts might be compensated for by use of AH.

Materials and Methods

All normal zygotes were maintained individually in micro-droplets of stage-appropriate culture medium until day-6 of development, when blastocysts were selected according to morphology and degree of expansion. An initial group of women (n=41) had blastocysts transferred with the ZP intact on day-6 (Control Group). A subsequent group of day-6 transfer women (n=39) were offered AH prior to transfer. In this Test Group all blastocysts were exposed to 0.1M sucrose in modified HTF for 1 minute to shrink the embryo, and a hole (35 to 40µm) was drilled with acidic Tyrode’s medium in the ZP away from the shrunken embryo. After washing, such AH blastocysts were allowed to re-expand prior to embryo transfer (ET, 1 to 4 hours later). Implantation rate (IR), clinical and ongoing pregnancy rates (CPR, OPR) were retrospectively compared after the transfer of day-6 blastocysts either with intact ZP or following ZP drilling.


Table 1 shows the outcomes relative to each study group:- following ET of an average of 2.6 blastocysts in the Control Group, CPR, OPR and IR were 29%, 22% and 14% respectively. This compared with the Test Group, in which all blastocysts underwent AH (average 2.4 for ET), where CPR, OPR and IR were 46%, 38.5% and 22.5% respectively. X2 analysis of these two day-6 Groups showed the following:- AH Test versus Control - CPR, p<0.05; OPR, p<0.1; IR, p<0.1. With blastocyst transfers with day-5 Controls in our program yielding CPR, OPR and IR of 58%, 55% and 32.5% respectively (significantly different from the day-6 Control Group; p<0.05), clearly day-6 Control blastocysts were performing worse, even though their morphology was often comparable with blastocysts transferred on day-5.


Day Six
Day Five
Assisted Hatching
age- years
embryos transferred
clinical preg. rate
ongoing preg. rate
implantation rate


Simplistically speaking, the fact that day-5 embryos seem consistently to outperform the day-6 embryos prior to the use of AH would seem to favor strongly the transfer of embryos on day-5, as if somehow the extended culture were deleterious in some way to the health of the embryo. This interpretation, however, limits the potential to maximize the selection of embryos at the blastocyst stage, in that not all embryos will have undergone blastulation by day-5. Leaving embryos until day-6 for ET can increase the overall population of blastocysts to choose from for transfer. With AH on day-6 as a tool to enhance implantation of these “slower” embryos, it should be possible to achieve improved pregnancy rates through improved selection. The results of this retrospective study suggest a distinct benefit from day-6 AH, with few of the concerns related to day-3 AH with premature opening of the ZP, and where rupture of blastomeres through the ZP hole may occur during traumatic ET. The improvement of implantation seen with the day-6 AH embryos is especially noteworthy since the AH group was both an older population of women who received a slightly lower number of embryos at ET. Interestingly, after day-6 AH many blastocysts were already hatching or fully escaped from their zonae at the time of ET. A prospective randomized study is currently being undertaken to attempt to confirm these results. Further, it is conceivable that laser drilling might further facilitate the ease with which blastocyst AH may be performed.



COHEN J. Zona pellucida micromanipulation and consequences for embryonic development and implantation. In Cohen J, Malter HE, Talansky BE & Grifo J (eds.), Micromanipulation of Human Gametes and Embryos, Raven Press, New York, pp.191-209, 1992.

FONG C-Y, BONGSO A, NG S-C, KUMAR J, TROUNSON A & RATNAM S. Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation. Hum Reprod, 13, 2926-32, 1998.

GARDNER DK, SCHOOLCRAFT WB, WAGLEY L, SCHLENKER T, STEVENS J & HESLA J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 13, 3434-40, 1998.

JONES GM & TROUNSON AO. Blastocyst stage transfer: pitfalls and benefits. The benefits of extended culture. Hum Reprod 14, 1405-8, 1999.

LIU HC, COHEN J, ALIKANI M, NOYES N & ROSENWAKS Z. Assisted hatching facilitates earlier implantation. Fertil Steril 60, 871-5, 1993.

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