Laser zona pellucida thinning—an alternative approach to assisted hatching

D.A. Blake1,3,3, A.S. Forsberg1, B.R. Johansson2 and M. Wikland1

1 Fertilitetscentrum, Carlander's Hospital, Göteborg, 2 Institute of Anatomy and Cell Biology, University of Göteborg, Sweden and 3 Glycoscience Research Centre, Auckland University of Technology, Auckland, New Zealand


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The purpose of this study was to assess the efficacy and hatching characteristics of in-vitro cultured human embryos subjected to laser zona pellucida thinning. METHOD: Zona thinning was performed on 110 embryos using a non-contact 1.48 µm diode laser and the hatch rate in vitro was compared with 42 control embryos. Variation of zona thickness and degree of zona expansion was assessed. Scanning electron microscopy was performed on embryos entrapped during hatching to identify the site of hatching. RESULTS: The rate of hatching was significantly higher in laser thinned blastocysts compared with control embryos (68 versus 33% respectively, P < 0.01). Laser thinning increased the variation of zona thickness in embryos from 11.6–27.3%. Natural zona thinning occurred in 92% of laser thinned hatching blastocysts and 100% of control embryos. CONCLUSION: These results suggest that laser zona thinning is effective and may provide significant advantages over conventional assisted hatching techniques, which create holes.

Key words: assisted hatching/in-vitro fertilization/laser/micromanipulation/zona pellucida


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
From the inception of IVF in 1978, pregnancy rates have risen slowly but steadily, to levels considered normal for the fertile population (HFEA 5th Annual Report, 1996Go; Lancaster et al.1997Go). However, the quest to break through this physiological ceiling continues, with much of the current emphasis now being placed on the events surrounding implantation.

There are three broad reasons for failure of implantation following replacement of apparently vital embryos. The first involves intrinsic embryonic factors that reflect slower development or deficiencies in the health of the blastocyst itself and its ability to hatch (Gott et al.1990Go; Plachot, 1992Go; van Kooij et al.1996Go). The second relates to extrinsic uterine factors that imply a lack of implantation receptivity in the endometrium (Edwards et al., 1986Go; Yaron et al.1994Go). Finally, failure of the embryo to escape the confines of the zona pellucida may be associated with a variety of clinical and laboratory circumstances (De Felici and Siracusa, 1982Go; Sharma et al.1990Go; Bertrand et al.1996Go). It is the latter reason that provides the rationale for the development of micromanipulative techniques aimed at assisting the hatching potential for in-vitro cultured embryos (Cohen et al.1990Go).

Assisted hatching has been performed since the early 1990s, with conflicting evidence being reported about the effectiveness of this procedure (Garrisi et al.1990Go; Cohen 1991Go; Hellebaut et al.1995Go). However, a meta-analysis of all published data reveals a significant clinical benefit of assisted hatching over controls (Brinsden et al.1997Go). The most commonly performed technique for assisted hatching is zona drilling with a stream of acid Tyrode's solution. Although this method has been reported to be largely effective in terms of pregnancies rates, it does present a number of practical disadvantages, including a risk of damage to the underlying cells, premature herniation and the substantial set-up time that is required. The latest addition to the armoury of assisted reproductive technology, the 1.48 µm diode laser Fertilase® (Medical Technologies Montreux SA, Clarens, Switzerland), now provides a precise and non-contact alternative that has overcome the drawbacks of previous methods (Germond et al., 1996Go).

The Fertilase® technology is relatively novel. Consequently there is little literature that exists describing the optimum hole size and laser duration (Rink et al., 1995Go). The conventional method for assisted hatching using Tyrode's solution or laser is to create a hole of ~20–40 µm that entirely perforates the zona. Initial studies in our clinic using this approach with excess embryos in extended culture suggested that a complete breach of the zona may be associated with several disadvantages. In a preliminary series of 26 embryos exposed to complete laser breach of the zona resulting in holes of 20–40 µm in diameter, eight of the nine resulting blastocysts showed signs of hatch initiation, which consisted of blastocoele herniation without associated natural zona thinning and embryo expansion (Figure 1AGo). Although four out of eight of these embryos were observed to complete the hatching process, there was concern at the high frequency of cellular remnants remaining post-hatching. Based on these observations, we decided to explore a less invasive approach of laser zona thinning.



View larger version (146K):
[in this window]
[in a new window]
 
Figure 1. (A) Embryo hatching through a laser induced hole of 30 µm in diameter. The embryo has herniated through the unexpanded zona of 16 µm in thickness. (B) Laser zona thinning. Six ablations on a duration setting of 15 ms were required to achieve this 80 µm region of thinning. (C) Embryo escaping centrally through a lasered region of the zona pellucida. The concave outline of the laser ablation is indicated by the arrow. (D) Scanning electron micrograph of a hatching site within the laser thinned region. The first laser ablation channel is indicated by the arrow.

 
Assisted zona thinning in both the human and animal model has been previously reported to provide a positive influence on hatching and implantation rates (Khalifa et al., 1992Go; Antinori et al., 1996Go). However, these studies report on the use of two types of contact methods, the YAG laser and cruciate thinning with Tyrode's solution, both of which substantially differ from the Fertilase® laser. It is clear from scanning electron microscopy that the architecture of the laser hole is different from that of the acid Tyrode's hole (Germond et al., 1995Go; Obruca et al., 1997Go). The tangential laser incision creates a trench in the zona with a central hole and it is possible that this pattern may result in unique hatching mechanisms. A key aspect of thinning that therefore requires authentication is the effect that it has on the embryo exit site.

Previous studies using videocinematography of IVF embryos prior to transfer have shown that variability of zona thickness within individual embryos is one of the most significant morphological predictive factors of implantation (Cohen et al., 1989Go). A recent study by Palmstierna and co-workers demonstrated that embryos with a zona thickness variation of >20% resulted in a 76% pregnancy rate with two embryos transferred (Palmstierna et al., 1998Go). These findings support the development of a technique such as laser thinning, whereby the variability of zona thickness is enhanced. It must however be emphasized that micromanipulation does not improve the intrinsic viability of an embryo, but merely attempts to give potentially less viable embryos assistance with their escape from the zona pellucida.

The aims of this study were to answer three key questions. Firstly, does laser thinning enhance embryo hatching in vitro? Secondly, what appearance does the laser thinned embryo have during hatching? And thirdly, is the site of laser thinning also the site of embryo hatching?


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study material and culture system
The study material consisted of embryos that were donated to research from consenting couples on completion of an IVF or intracytoplasmic sperm injection (ICSI) embryo transfer between March and September 1998. These represented embryos that had failed the laboratory's morphological selection criteria for embryo transfer and freezing on day 2 or 3 post-oocyte retrieval. Swedish law permits the use of human embryos in excess of IVF cycles to be used for experimentation provided the couple has given written consent to such experimentation. Thus according to Swedish law all couples included in this study gave written consent to experimentation on their excess embryos at the time of signing routine consent forms for IVF on the first day of each stimulated cycle. All control and experimental embryos were fixed in glutaraldehyde by the 7th day of embryonic development.

Due to the limited supply of experimental material and the need to gain as much experience as possible with the thinning technique, a 2:1 allocation of embryos into the experimental and control group was applied. The study design, although not fully randomized, was prospective and attempts were made to be as unbiased as possible during the allocation of embryos to the two groups on day 3 of development.

Embryos were pooled in groups of up to four, in 50 µl drops of IVF-50® (IVF Science, Göteborg, Sweden), overlaid with mineral oil (Ovoil®; IVF Science) and cultured in a 37°C humidified incubator (Cytoperm®; Heraeus, Upplands Väsby, Sweden) at 5% CO2/air until day 3. Embryos that had developed to the 6- to 8-cell stage by 78 h were included in the study. Laser assisted hatching was performed between 1600 and 1730 h on day 3 and the embryos were washed in two drops of fresh media before being placed in 0.7 ml S2 media (IVF Science) in a Petri dish (Falcon 3037; Becton Dickinson, NJ, USA) and cultured until day 7. The S2 media was replenished every 48 h with freshly incubated media.

Laser intervention
Laser manipulations were performed using a 1.48 µm (infrared) diode laser (Fertilase®) and a 640 µm pilot light on an inverted microscope with heated stage (Diaphot 300; Nikon). The pilot light was calibrated to a target on a video monitor (PVM 96E; Sony, Tokyo, Japan) which was used as the primary visualization mode for aiming the laser during use (i.e. not via the microscope eyepiece). Prior to manipulation each embryo was randomly positioned and assessed for variation of zona thickness by measuring the zona at three points (9, 12 and 3 o'clock) using digital callipers (Viacontroller VIA-100; Boeckeler Instruments Inc., AZ, USA) on the monitor. This method of measurement has been previously described by others (Cohen et al., 1989Go; Palmstierna et al., 1998Go). The precision of the zona measurements was 0.5 µm (2.9%).

Thinning
Experimental embryos were positioned on the microscope stage with the laser target located on the outer edge of the zona. A maximum of six ablations were made successively around the zona to achieve a depth of 50–80% of the zona thickness for a total length of approximately 80 µm (Figure 1BGo).

Control
A control group of embryos underwent a sham laser exposure, equivalent to the experimental group. The laser beam was positioned 100 µm away from the control embryos so that no zona disruption occurred.

Assessment
Variation in zona thickness was calculated by the following formula (Cohen et al., 1989Go):


From day 5, embryos were assessed daily between 1600 and 1700 h on an inverted microscope with heated stage for a maximum of 2 min per culture dish. Photographic documentation was recorded on a digital recorder database (Sony Colour Video Camera DXC-151P, Sony Video Cassette Recorder GV-D900E, Sony Digital Recorder DKR-700P; Sony). Evidence of blastulation, blastocoele expansion (including zona thickness and total embryo diameter), stage of hatching and exit site of hatching was recorded. Hatch initiation describes all embryos that show signs of cellular extrusion outside the external surface of the zona. Embryos that had not completely hatched by day 7 of embryonic development were considered trapped. Complete hatching describes embryos where the zona was entirely detached and separate from the embryo. Embryos showing no signs of further development between day 5 and 6 were fixed on day 6, while all remaining embryos were fixed on day 7.

Scanning electron microscopy
Embryos were fixed in 2.5% glutaraldehyde in 0.05 mol/l Na cacodylate buffer, pH 7.2 for 2–24 h. The embryos were washed extensively in phosphate buffered saline (PBS) and placed onto the plastic coverslips (Thermanox®) in 2 µl droplets of PBS. The coverslips had been pre-coated with gold in a sputter coater and treated with 20 µl droplets of 1% poly-L-lysine that were allowed to dry immediately before use. The embryos were post-fixed and made electrically conductive with the OTOTO method (repeated treatments with 1% OsO4 and a saturated solution of thiocarbhydrazide; Friedman, 1981), followed by dehydration in a graded series of ethanol. The alcohol was substituted with hexamethydisilazane, which was allowed to evaporate. The resulting dried specimens were mounted on aluminium stubs and were examined in a Zeiss 982 Gemini field emission scanning electron microscope (Zeiss, Oberkochen, Germany) at a working distance of 3–4 mm and an accelerating voltage of 2–3 kV.

Statistics
The normality of data was tested using the Ryan–Joiner test. Differences between mean zona thickness and differences in variability were assessed using the Student's t-test (independent and paired). The {chi}2 test was used to test if the rate of hatching was independent of the thinning treatment.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
All thickness variables were consistent with assuming normality. There was no statistical difference between the experimental and control group prior to laser thinning for mean zona thickness (17.1 µm versus 16.2 µm) and mean variation of thickness (11.6 versus 11.7%). Laser thinning was effective in increasing the variation of zona thickness (from 11.6–27.3%, P < 0.0001, in the experimental group).

There was no statistical difference in the blastulation rate (Table IGo) between the experimental and control group (P = 0.597). Significantly more blastocysts initiated hatching in the experimental group compared with the control group (P = 0.009). None of the control group (0/6) completed hatching within 7 days of culture compared to 50% (19/38) of the thinned group.


View this table:
[in this window]
[in a new window]
 
Table I. Comparative rates of initial hatching, expanded hatching, embryo entrapment, and complete hatching in laser zona-thinned and control embryos
 
Of the 38 blastocysts that initiated hatching in the experimental group, positive identification of a laser hatch site was made in 16 embryos. The remaining embryos had hatch sites that were either unidentifiable or not recorded due to inadequate orientation or attrition during the SEM processing. There were no reports of embryos hatching from sites other than the laser site. Exit sites were located both central and peripheral to the lasered area (Figures 1C and DGo). The architecture of the laser cuts with the thinning technique consisted of multiple channels that covered a total surface area of approximately 40x60 µm (Figure 1DGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
To date there are a limited number of studies that describe the in-vitro development and hatching process of zona manipulated human embryos (Dokras et al., 1994Go; Nakayama et al., 1998Go). While these investigations may not be representative of the hatching mechanisms in vivo, they do provide us with the ability to compare the effect of different interventions to the zona. Our initial observations of zona escape from embryos that had undergone assisted hatching through the creation of laser holes provided us with concern. Although these embryos hatch earlier as intended, they do so by a slow herniation through a small hole in what remains a thick zona. This process may create a danger for the embryo to be damaged or split, as has been described as the cause in cases of monozygotic twinning in micromanipulated embryos (Cohen et al., 1990Go; Alikani et al., 1994Go). It was primarily these concerns that led us to investigate an alternative method for laser assisted hatching and the development of zona thinning.

One of the reasons that there are so few studies assessing human hatching mechanisms is due to the difficulty in obtaining adequate quantities of study material. The quantity of excess embryos available for research is dependent on a number of factors, such as a country's research regulations, the clinics' freezing criteria, and the proportion of patients consenting to research. More importantly, the quality of excess embryos is inherently low. It is because of this high attrition rate that embryos were recruited into this study on day 3 when they had demonstrated continuous cleavage past the 4-cell stage. The relatively high blastulation rate (43–51%) seen in this study is testament to the stringent selection process of embryos on day 3. There was no significant difference in blastulation rate between the experiment and control group.

In this study, almost twice as many zona-thinned embryos initiated hatching as in the control group. The rate of hatch initiation and completion in the non-manipulated embryos is similar to other reports (Dokras et al., 1991Go; Nakayama et al., 1998Go). Complete hatching, while not guaranteed with zona thinning, occurred at a higher rate than controls. It is nevertheless possible that complete hatching may have occurred if the cut-off point for the study had been later than day 7. Due to the small number of embryos in the control group the results of the statistical analysis were unreliable for complete hatching rates, however there was sufficient evidence to suggest that a level of significance would be reached with a larger sample. But perhaps the most significant observation was the degree of blastocyst expansion achieved in zona thinned embryos during development. By day 6 all thinned blastocysts had displayed some degree of expansion and natural zona thinning to <10 µm. In contrast, no blastocyst expansion was observed in our preliminary observations of embryos receiving a complete laser breach of the zona, an observation that has been described elsewhere (Cohen et al., 1990Go; Cohen and Feldberg, 1991).

It is important to note that despite years of investigation, the exact mechanism of zona escape in vivo has yet to be elucidated. Perhaps the most confusing aspect of this area of research is the high degree of interspecies variability. Zona escape in the hamster appears to be independent of the embryo with the expression of an endocrine-controlled protease secretion in the pregnant uterus (Gonzales and Bavister, 1995Go). In contrast, cattle embryo hatching appears to be independent of lysins with blastocysts being regularly flushed from the uterus in a hatched state with accompanying empty zona (Massip, 1982Go). Temporal measurements of the total embryo volume and zona thickness in cattle embryos during embryo expansion suggests that thinning is a mere consequence of internal pressure (Schmoll and Schneider, 1999Go).

It could be that the characteristic hatching process observed in vitro, i.e. blastocoele expansion associated with zona thinning, is in fact an artefact. One argument is that the so-called `bursting' out of the zona that is seen in vitro may be a fall back mechanism that operates in the absence of uterine proteases (Gunnell and Ewings, 1994Go). Alternatively, it may be possible that culture conditions, including media constituents, may play an understated role in the disparity of results reported in both in-vitro hatching studies and human assisted hatching clinical trials. For example, the presence of plasminogen in serum or crude bovine serum albumin has recently been reported to interact with embryo-derived urokinase-type plasminogen activator which is hypothesized to be involved in the hatching process (Menino et al., 1997Go). It is possible that differences in the protein supplementation of culture media may account for the substantial improvement in some clinics' pregnancy rates with assisted hatching, while others using identical manipulation techniques experience no benefit.

Zona thinning is not a new concept, but what is novel to this study is the method. It has previously been shown in mouse embryos (Khalifa et al., 1992Go) that cruciate zona thinning with Tyrode's solution produced earlier and higher complete hatching rates than zona hole drilled controls. These zona-thinned embryos also resulted in a significantly lower arrest rate at the morula stage than controls. Using this method of thinning, Tucker and co-workers failed to show a significant improvement in human implantation rates, but suggested that it may have had a positive influence on a subgroup of embryos exhibiting a small variation of zona thickness (Tucker et al.1993Go). The first reports of laser zona thinning in a human series were published in 1996 (Antinori et al.1996Go). Using a YAG laser, 50% of the zona thickness was thinned for a length of 20 µm on day 2. In this paper, the authors did demonstrate a statistically significant increase in implantation rate in a large heterogeneous patient population when compared with matched controls.

At least two published studies to date have demonstrated the relationship between highly variable zona thickness and high pregnancy rates (Cohen et al.1989Go; Palmstierna et al.1998Go). Laser thinning of a substantial area of the zona may serve to enhance this relationship, particularly for embryos with invariable zona thickness. It has been stated that overall zona thickness varied between age groups and types of infertility (Loret De Mola et al.1997Go). Therefore it is possible that laser thinning could also be of benefit for a variety of indications such as patients over the age of 38 years (Brinsden et al.1997Go), unexplained infertility (Loret De Mola et al.1997Go) and low embryo quality.

In the development of this technique particular care was taken to penetrate the inner dense layer of the zona, without which the laser ablation may be little more than cosmetic. Unfortunately, control of the depth of ablation is one of the most variable aspects of this technique with respect to operator judgement and laser precision. Ablation of the zona at this wavelength (1.48 µm) results in a relatively unpredictable hole size which may be dependent on the water content of the glycoprotein matrix. Is was therefore difficult to quantify this aspect of the technique with accuracy except to say that penetration into the darker inner layer of the zona was achieved without complete perforation. In 42% of hatching blastocysts in the experimental group, it was possible clearly to visualize the hatch site. In all of these cases, the embryo was positively identified as escaping via the laser thinned area. Scanning electron microscopy revealed a tear centrally located through the laser thinned domain, suggesting that this technique creates a natural line of weakness.

A theoretical drawback of zona thinning is that the increased number of laser exposures required to create a large gap may be detrimental to the embryo. However, none of the observations during this study gave evidence to back up this theory. Indeed experimental embryos developed at an equal rate to control embryos. Conversely there is less risk of blastomere damage with zona thinning due to the laser beam being positioned further away from the cells than techniques for complete zona breach. Studies to date have shown Fertilase®-induced zona holes to be a safe modality of treatment, with respect to embryo development and fetal outcome in mice (Germond et al.1995Go).

This study demonstrates that laser thinning significantly increases the hatching and complete hatching rates in vitro, compared with control embryos. By not breaching the zona, we avoid the potential risk of blastomere loss, embryonic infection and the subsequent prophylactic administration of immunosuppressants and antibiotics. Embryo expansion and natural zona thinning is observed in all zona thinned blastocysts, thus preserving normal physiological development prior to hatching. Adjustments to the variation of zona thickness after laser zona thinning may aid in transforming embryos from a low to a high pregnancy prognosis group. A prospective randomized trial is now needed to test this hypothesis.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank the Fertilitetscentrum laboratory staff for their technical support and Drs Margareta Wood and Torbjörn Hillensjö for advice during this study. The authors also appreciate the statistical assistance provided by Neil Binnie in the Department of Applied Maths at Auckland University of Technology.


    Notes
 
3 To whom correspondence should be addressed at: Glycoscience Research Centre, Auckland University of Technology, Private Bag 92006, Auckland 1020, New Zealand. Email: debbie.blake{at}aut.ac.nz Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Alikani, M., Noyes, N., Cohen, J. et al. (1994) Monozygotic twinning in the human is associated with the zona pellucida architecture. Hum. Reprod., 9, 1318–1321.[Abstract]

Antinori, S., Panci, C., Selman, H.A. et al. (1996) Zona thinning with the use of laser: a new approach to assisted hatching in human embryos. Hum. Reprod., 11, 590–594.[Abstract]

Bertrand, E., Van den Bergh, M. and Englert, Y. (1996) Clinical parameters influencing human zona pellucida thickness. Fertil. Steril., 66, 408–411.[ISI][Medline]

Brinsden, P., Mascarenhas, L. and Avery, S. (1997) Clinical benefits of assisted hatching. In Gomel, V. and Leung, P.C.K. (eds) IVF and assisted reproduction. Monduzzi Editore, pp. 621–626.

Cohen, J. (1991) Assisted hatching of human embryos. J. In Vitro Fert. Embryo Trans., 8, 179–190.[ISI][Medline]

Cohen, J. and Felberg, D. (1991) Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo. Mol. Reprod. Dev., 30, 70–78.[ISI][Medline]

Cohen, J., Wiker, S.R., Inge, K.L. et al. (1989) Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil. Steril., 51, 821–827.

Cohen, J., Elsner, C., Kort, H.M.H. et al. (1990) Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum. Reprod., 5, 7–13.[ISI][Medline]

De Felici, M. and Siracusa, G. (1982) Spontaneous hardening of the zona pellucida of mouse oocytes during in vitro culture. Gam. Res., 6, 107–113.[ISI]

Dokras, A., Sargent, I.L., Ross, C. et al. (1991) The human blastocyst: morphology and human chorionic gonadotrophin secretion in vitro. Hum. Reprod., 6, 1143–1151.[Abstract]

Dokras, A., Ross, C., Gosden, B. et al. (1994) Micromanipulation of human embryos to assist hatching. Fertil. Steril., 61, 514–520.[ISI][Medline]

Edwards, R.G., Fishel, S.B., Cohen, J. et al. (1986) Factors influencing the success of in-vitro fertilisation for alleviating human infertility. J. In Vitro Fert. Embryo Trans., 1, 3–23.

Friedman, P.L. and Ellisman, M.H. (1981) Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnation. J. Neurocytol., 10, 111–131.[ISI][Medline]

Garrisi, G.J., Talansky, B.E., Grunfeld, L. et al. (1990) Clinical evaluation of three approaches to micromanipulation-assisted fertilization. Fertil. Steril., 54, 671–677.[ISI][Medline]

Germond, M., Nocera, D., Senn, A. et al. (1995) Microdissection of mouse and human zona pellucida using a 1. 48 µm diode laser beam: efficacy and safety of the procedure. Fertil. Steril., 64, 604–611.[ISI][Medline]

Germond, M., Nocera, D., Senn, A. et al. (1996) Improved fertilization and implantation rates after non touch zona pellucida microdrilling of mouse oocytes with a 1.48 µm diode laser beam. Hum. Reprod., 11, 1043–1048.[Abstract]

Gonzales, D.S. and Bavister, B.D. (1995) Zona pellucida escape by hamster blastocysts in vitro is delayed and morphologically different compared with zona escape in vivo. Biol. Reprod., 52, 470–480.[Abstract]

Gott, A.L., Hardy, K., Winston, R.M.L. et al. (1990) Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos. Hum. Reprod., 5, 104–108.[Abstract]

Gunnell, D.J. and Ewings, P. (1994) Infertility prevalence needs assessment and purchasing. J. Pub. Health Med., 16, 29–35.[Abstract]

Hellebaut, S., De Sutter, P., Onghena, A. et al. (1995) Does assisted hatching improve implantation rates after IVF or ICSI in all patients: a prospective randomized study. J. Assist. Reprod. Genet., 12, 107S.

HFEA 5th Annual Report (1996) Human Fertilisation and Embryology Authority: American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology: Assisted Reproductive Technology in the United States and Canada: 1994 results generated from the American Society of Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil. Steril., 66, 697–705.[ISI][Medline]

Khalifa, E.A.M., Tucker, M.J., and Hunt, P. (1992) Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse. Hum. Reprod., 7, 532–536.[Abstract]

Lancaster, P., Shafir, E., Hurst, T. et al. (1997) Assisted conception Australia and New Zealand 1994 and 1995. Australian Institute of Health and Welfare National Perinatal Statistics Unit and Fertility Society of Australia.

Loret De Mola, J.R., Garside, W.T., Bucci, J. et al. (1997) Analysis of the human zona pellucida during culture; correlation with diagnosis and the preovulatory hormonal environment. Assist. Reprod. Genet., 14, 332–336.

Massip, A. (1982) The behaviour of cow blastocysts in vitro: cinematographic and morphometric analysis. J. Anat., 134, 399–405.[ISI][Medline]

Menino, A.R. Jr, Hogan, A., Schultz, G.A. et al. (1997) Expression of proteinases and proteinase inhibitors during embryo-uterine contact in the pig. Devel. Gen., 21, 68–74.

Nakayama, T., Fujiwara, H., Tastumi, K. et al. (1998) A new assisted hatching technique using piezo-micromanipulator. Fertil. Steril., 69, 754–788.

Obruca, A., Strohmer, H., Blaschitz, A. et al. (1997) Ultrastructure observations in human oocytes and preimplantation embryos after zona opening using an erbium-yttrium-aluminium-garnet (Er;YAG) laser. Hum. Reprod., 12, 2242–2245.[Abstract]

Palmstierna, M., Murkes, D., Csemiczdy, G. et al. (1998) Zona pellucida thickness variation and occurrence of visible mononucleated blastomeres in preembryos are associated with a high pregnancy rate in IVF treatments. J. Assist. Reprod. Genet., 15, 70–75.[ISI][Medline]

Plachot, M. (1992) Viability of preimplantation embryos. Baillière's Clin. Obst. Gynaecol., 6, 327–338.

Rink, K., Descloux, L., Delacretaz, G. et al. (1995) Zona pellucida drilling by a 1.48 µm laser: influence on the biomechanics of the hatching process. SPIE Barcelona Proceedings, p. 2624.

Schmoll, F. and Schneider, H. (1999) Zona pellucida thinning during expansion of in vitro produced cattle blastocysts. Reprod. Dom. Anim., 34, 11.

Sharma, V., Whitehead, M., Mason, B. et al. (1990) Influence of superovulation on endometrial and embryonic development. Fertil. Steril., 53, 822–829.[ISI][Medline]

Tucker, M.J., Luecke, N.M, Wiker, S.R. and Wright, G. (1993) Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation. J. Assist. Reprod. Genet., 10, 187–191.[ISI][Medline]

van Kooij, R.J., Looman, C.W., Habbema, J.D. et al. (1996) Age-dependent decrease in embryo implantation rate after in vitro fertilization. Fertil. Steril., 66, 769–775.[ISI][Medline]

Yaron, Y., Botchan, A., Amit, A. et al. (1994) Endometrial receptivity in the light of modern assisted reproductive technologies. Fertil. Steril., 62, 225–232.[ISI][Medline]

Submitted on December 15, 2000; accepted on June 5, 2001.