Re: Relevance of the site of assisted hatching in thawed human blastocysts

16 06 2010

To the Editor:

With interest we read the elegant paper of Miyata et al. (1) which reported the existence of polarity in the hatching process of warmed human blastocysts. In detail, artificial zona pellucida (ZP) opening close to the inner cell mass (ICM) resulted in improved rates of complete hatching, whereas assisted hatching at the abembryonic site caused trapping of the embryo within the ZP (1).

This finding is completely in line with previously published data on the implantation behavior of fresh blastocysts showing different sites of herniation (2). In this manuscript (2), a significantly higher clinical pregnancy rate (72%) was observed if blastocysts were transferred that hatched close to the ICM as compared to their counterparts that herniated from the mural trophectoderm (51%). This (2) suggests that hatching problems observed during vitrification/warming (1) may be overcome in fresh blastocyst culture.

Spontaneous hatching of the human blastocyst in vitro is supported by the increase of internal pressure caused by a gradual accumulation of blastocoelic fluid and cellular (mostly trophectodermal) proliferation. Additional mechanical help to open the outer shell comes from sphincter-like zona-breaker cells (3) and sequences of repeated collapses and re-expansions (“blastocyst breathing”).

It is likely that in vitrified and warmed blastocysts complete opening of the ZP by means of laser pulses (1; Figure 1A and 1B) changes the pressure conditions, which would be one of the prerequisites for optimal hatching. If the function of zona-breaker cells is also negatively affected by manipulation of the ZP, the hatching process per se cannot be actively completed and, theoretically, in a more passive mode has to follow physical rules, such as the Hagen-Poiseuille equation (1). Thus, a blastocyst hatching close to the ICM, a location corresponding to the area that later drives invasion into the endometrium, has accelerated contact to the endometrium which could result in a developmental advantage, irrespective of whether it is fresh or cryopreserved.

Thomas Ebner, Ph.D.
Omar Shebl, M.D.
Richard B Mayer, M.D.
Gernot Tews, M.D.
Landes-Frauen- und Kinderklink
IVF Unit
Linz, Austria

References
1. Miyata H, Matsubayashi H, Fukutomi N, Matsuba J, Koizumi A, Tomiyama T. Relevance of the site of assisted hatching in thawed human blastocysts: a preliminary report. Fertil Steril 2010; in press.

2. Ebner T, Gruber I, Moser M. Location of herniation predicts implantation behaviour of hatching blastocysts. J Turkish-German Gynecol Assoc 2007; 8: 184-8.

3. Sathananthan H, Menezes J, Gunasheela S. Mechanisms of human blastocyst hatching in vitro. Reprod Biomed Online 2003; 7: 228-34.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.06.026

The Authors Respond:

We would like to express our thanks to Ebner et al. for their interest in our paper on the existence of polarity in the hatching process of thawed human blastocysts (1).

Our data from a prospective randomized study of assisted hatching (AH) of ‘thawed’ human blastocysts (N=32) demonstrated that the rate of complete hatching was significantly higher for AH performed near the ICM than for AH at the side opposite to the ICM (1). In ‘thawed’ blastocysts without AH (N=13), initiation of hatching occurred almost equally at sites near and opposite the ICM. Extrusion of the ICM and complete hatching, however, were only observed near the ICM (1). Our study had limitations of a small sample size, the use of frozen embryos, and the in vitro nature of the study. Moreover, we did not know whether pregnancy or implantation would be related to the hatching site, because we did not attempt any transfer of the studied blastocysts. Ebner’s paper (2) seemed to compensate for these limitations. Using ‘fresh’ blastocysts without AH (N=108), they reported that a significantly higher clinical pregnancy rate was observed if blastocysts that hatched close to the ICM were transferred as compared to their counterparts that herniated from the mural trophectoderm (2). This finding strengthens the evidence supporting the existence of polarity in the hatching process of human blastocysts.

We completely agree with their discussion. The mechanisms of human blastocyst hatching in vitro require: 1) increased internal pressure; 2) sphincter-like zona-breaker cells (3); and 3) sequentially repeated collapses and reexpansions (blastocyst breathing). Therefore, in our study, AH at the side opposite the ICM may: 1) change the internal pressure prerequisites for optimal hatching; 2) break the zona-breaker cells; 3) weaken blastocyst breathing, resulting in a more passive mode such as the Hagen-Poiseuille equation indicated in our paper (1). As Ebner et al. also indicated in their paper (2), these proposals are still theoretical. Whether these truly are the mechanisms involved in the human hatching process and whether some of these mechanisms are disrupted during AH require further study.

Finally, it should be emphasized that the blastocysts studied were all from infertile patients and that the data were in vitro. We must consider that these data may not reflect the ‘normal’ behavior of blastocysts hatching ‘in vivo’. Our and their findings, however, suggest that some AH procedures may assist while other AH procedures may inhibit hatching because of the existence of polarity in the hatching process. At least, these findings direct physicians’ or embryologists’ attention to the choice of AH site.

Hirotoshi Miyata A.S.
Hidehiko Matsubayashi M.D.
Noriko Fukutomi A.S.
Junko Matsuba B.S.
Azusa Koizumi A.S.
Tatsuhiro Tomiyama M.D.
Osaka New ART Clinic and New ART Research Center
Osaka, Japan

References
1. Miyata H, Matsubayashi H, Fukutomi N, Matsuba J, Koizumi A, Tomiyama T. Relevance of the site of assisted hatching in thawed human blastocysts: a preliminary report. Fertil Steril 2010; in press.
2. Ebner T, Gruber I, Moser M. Location of herniation predicts implantation behaviour of hatching blastocysts. J Turkish-German Gynecol Assoc 2007; 8: 184-8.
3. Sathananthan H, Menezes J, Gunasheela S. Mechanisms of human blastocyst hatching in vitro. Reprod Biomed Online 2003; 7: 22

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.06.025

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