London Gynaecology and Fertility Centre, London, W1G 7JH UK
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Abstract |
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Key words: Assisted hatching/embryos/IVF/implantation/laser assisted hatching
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Introduction |
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One of the problems with assisted hatching has been the requirement of extensive technical skill to produce uniform and standardized holes using an acid solution with micro tools, operated under the control of micromanipulators. The drilling of the ZP by laser has been proposed as an alternative to these traditional methods of assisted hatching (Tadir et al., 1991).
Laser and light delivery systems have significant advantages over the chemical or mechanical drilling procedures in that they provide touch-free objective-delivered accessibility of laser light to the target, with minimal absorption by the embryos (Germond et al., 1995). Secondly, the lasers now used for this purpose are affordable and easily adapted to any existing inverted microscope. Furthermore, the laser target in the reaction process is controlled accurately and has been shown to produce the opening in the ZP with no mechanical, thermal or mutagenic side effects (Germond et al., 1995
). On this basis, and after the licence for laser assisted hatching was granted to our centre by the Human Fertilisation and Embryology Authority (HFEA), we introduced laser assisted hatching in the UK. Our initial results using the laser to create a single hole completely through the ZP were disappointing. We therefore modified our laser technique to thinning the zona in one particular area. This small change seemed to improve our results significantly. Following this development, and bearing in mind the changes that normally happen in the natural, non-assisted hatching blastocyst process, we speculated that laser application to an extended area of the ZP, rather than at a point, would perhaps further improve our pregnancy rates. Since the site along the zona where natural hatching is likely to take place cannot easily be identified, increasing area of zona thinning was thought to encompass these sites and facilitate this process. In this paper, we present a retrospective analysis of our treatment results comparing the three types of laser assisted hatching (LAH).
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Materials and Methods |
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All patients received written information about laser-assisted hatching and were given an opportunity to discuss this technique with their clinician before signing the relevant consent form.
Total LAH was performed on embryos from 77 patients who fitted in one or more of the above categories. Embryos from 158 patients underwent partial assisted hatching with a further 87 patients embryos undergoing quarter LAH. Patients who agreed to LAH underwent routine IVF/ICSI treatment cycle procedures in order to generate embryos for transfer. The stimulation protocols for IVF and ICSI have already been previously outlined (Meniru and Craft, 1997; Craft et al., 1999
). No patients undergoing a frozen embryo replacement were included in this study.
On day 1 of embryo culture, assessment of fertilization was performed and only those embryos exhibiting 2 definite and clear pronuclei were replaced in a fresh culture well of a 4-well multidish (Nunclon, Denmark) containing 0.5 ml of IVF-50 medium (IVF Science, Sweden) overlaid with 0.4 ml of liquid paraffin oil (IVF Science, Sweden). Approximately 15 embryos per well were cultured together for a further 24-hours at 37°C in an incubator with an atmosphere of 5% CO2 in air.
On the morning of day 2, the embryos were scored, and up to 3 embryos deemed suitable for transfer were selected for assisted hatching. It was preferable that the embryos selected for transfer had a minimum cumulative embryo score of 12, i.e. 4 cells with a grading of at least 3 out of 4, or 3 cells with 4 out of 4 grading. The cumulative embryo score was obtained by multiplying the number of cells with the embryo grade (grade 14), which was based on the degree of fragmentation. An embryo with a grading of 4 showed no fragmentation, whereas a grade 3 embryo contained up to 25% fragmentation. Any remaining embryos of similar quality were frozen. These supernumerary embryos were frozen intact and not drilled using the laser. Embryos deemed unsuitable for cryopreservation, did not undergo assisted hatching and were discarded. Embryos that were deemed suitable for transfer, and had preferably divided overnight in culture, were eligible for laser hatching. Fourteen days post embryo transfer patients undertook a pregnancy blood test. Only women with a ßHCG >25 IU were considered positive. At 7 weeks gestation a clinical pregnancy was established with the presence of a fetal heart beat.
Microsurgical laser hatching
Due to the specific wavelength used, laser drilling was performed directly on the embryos in the 4-well multidish (Nunclon), therefore keeping the embryos in their original culture medium. The dish was placed onto the displacement stage of the diaphot inverted microscope (Nikon, Japan) fitted with a Fertilase micro drill (FertilaseTM , Medical Technologies, Montreux SA, Switzerland). The set up used for ZP microdrilling was similar to that described in detail elsewhere (Rink et al., 1994; Germond et al., 1995
). Briefly, the laser micro-surgical system consisted of an invisible laser diode beam emitting at a wavelength of 1.48 µm (surgical laser), which was collimated and matched with a 1 mW visible 670 nanometer diode laser aiming beam. These beams were fed into the inverted microscope via the fluorescent port through several mirrors and focused by a 40x microscope objective. The beam focusing through the microscope led to a measured spot size (~810 µm) that was magnified and observed on an external monitor. Laser drilling was achieved in either one or two irradiations at a typical power of 47 mW and, for human embryos, an exposure time of 20 ms was required. Changing the irradiation time could control the size of the generated aperture. Under these conditions however, the exposure to the laser light was short enough to ensure precise and localized lysis of the ZP, and therefore it was not necessary to hold each embryo with a suction pipette.
With the help of the displacement stage, a region of the ZP where the perivitelline space was widest was positioned at the location of the aiming spot. The diameter of the ZP covered ~30 to 40% of the screen monitor, thus ensuring the aiming spot could be accurately placed between the middle and outer edge of the ZP. Exposing the ZP to laser light was achieved by using a foot pedal to control the switch. The irradiation time of 20 ms allowed a hole to be drilled the size of half the zona. The centre of the non-drilled portion of the ZP was then moved to the position of the aiming spot on the monitor, and a second hole was laser drilled. This ensured the inner membrane was broken and a clear hole through the ZP was created: total LAH (Figure 1). The drilled hole was ~1.5 times the thickness of the ZP. Positioning of the embryos was such that there was a clear space between the blastomeres and the ZP to ensure that the cytoplasm was not touched by the laser. Once the embryos were drilled, they were placed back into the incubator until the transfer procedure.
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Results |
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Discussion |
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Our results strongly suggest that the outcome of LAH is dependent on the mode by which the ZP is breached, as well as the size of the artificial gap. Total LAH was introduced first in our centre based on the literature and information presented on LAH at that time (Germond et al., 1995, 1996
, 1998
; Antinori et al., 1996
). It has also been shown that zona thinning alone is not sufficient in promoting implantation, suggesting the inner layer of the human ZP has to be fully breached (Tucker et al., 1993
). The pregnancy and clinical pregnancy rates in this group of patients did not fulfil our expectations. The pregnancy rate in this group was 14.6% and subsequent clinical pregnancy rate dropped to 5.2%. The possible explanations for this relatively bad outcome may have been due to: (i) the breaching of the inner membrane and rapid exposure of the embryos to cells of the immune system. Although patients in this group were administered a low dose of an immunosuppressant to reduce the presence of white cells and any local inflammatory response that might have occurred at the time of embryo transfer, embryos were hatched on day 2 of development thus being exposed to the uterine surface for a longer period of time. Previous studies have suggested that the risk of immunosuppressants at a low dose is not harmful to the embryo (Cohen et al., 1990
), (ii) it may be that total laser assisted hatched embryos were at a higher risk of losing blastomeres through the hole as the hatching was performed prior to compaction. (iii) Alternatively, it may be those total LAH drilled embryos were at greater risk of becoming trapped. Published results (Cohen and Feldberg, 1991) showed small holes resulting from application of mechanical zona dissection, inhibited completion of the hatching process rather than improving implantation rates; (iv) using the laser as opposed to traditional chemical or mechanical means of assisted hatching may well be altering the ZP in a different way such that the effect of breaching the inner layer of the zona is detrimental to continued normal development and hatching of the embryo; (v) sudden change in biochemical environment without time for adaptive changes and (vi) finally, detrimental effects of the laser itself although as has been previously suggested (Germond et al., 1995
) that the laser had no mechanical thermal or mutagenic effect on the embryos, in view of our results this may not be the case for total LAH.
To try and negate some of these possibilities for the poor outcome of the total LAH, partial LAH was subsequently performed which involved thinning the ZP and not breaching its inner membrane. No steroids or antibiotics were given to this group of patients as the zona was effectively weakened but remained intact. Furthermore, there was no direct hole for blastomeres to potentially escape from or become entrapped. A significant improvement in pregnancy rate (20.9%; P < 0.001) and clinical pregnancy rate (18.3%; P < 0.001) was obtained in comparison with the total LAH group. Quarter LAH was thought to mimic the physiological process of hatching by creating a greater area of weakened ZP. A significant increase in pregnancy rate (29.0%; P < 0.05) and clinical pregnancy rate of (22.1%; P < 0.05) was also achieved when compared with the total LAH group.
Although an earlier report suggested a total breach of the human zona was important for successful hatching (Tucker et al., 1993), it may be that the laser has a different effect on the zona in comparison with chemical methods for hatching, particularly if carried out on a human embryo on day 2 of development. Our results suggest that the breaching of the inner zona layer using the laser is detrimental to the implantation potential of the human embryo. Previous data (Khalifa, 1992) observed a beneficial effect on mouse embryo hatching when a wide area of the outside layer of the ZP was removed, without piercing the glycoprotein matrix completely. It may be that expanding the number of breaking points over a larger area of the ZP is more likely to coincide with the site along the zona where natural hatching is likely to take place.
The differences in miscarriage rates amongst the three groups did not carry statistical significance. However, the large proportion of patients in the total LAH group, who miscarried prior to their ultrasound scans performed at 7 weeks, was of great concern. This seemed to indicate problems with either an incomplete implantation process or poor embryonic development as a result of the total LAH. Whether this was directly due to the total breach of the ZP or other factors associated with the laser is not certain.
The incidence of multiple pregnancies was also considered to be higher than expected, considering the type of patients that were offered this treatment. Whilst the sample size of patients was small, a multiple pregnancy rate of 23.3% in the partial LAH and 27.7% in the quarter LAH group also raised concerns. A similar observation has been made by other authors (Check et al., 1996) suggesting that the occurrence of monozygotic twins may be higher in patients undergoing LAH.
There is considerable variation in the results achieved with assisted hatching between different groups, almost certainly due to the differences in the technical procedures used and also influenced by differences in patient selection for the procedure. With the use of an acid solution, it is difficult to achieve the same degree of uniformity amongst different operators and various situations, and the chemical needs to be washed out to prevent any damage to the embryo. The diode laser offers many advantages over traditional methods in a clinical situation where the ZP needs to be opened. It is also evident that the further studies are needed to fully appreciate the effects of the laser on human embryo development and assess which patients might benefit from assisted hatching. We would like to address this issue with further clinical studies, particularly to assess the effect of LAH in more favourable groups having their first IVF/ICSI. Current protocols are being established to undertake a randomized prospective study comparing women with similar fertility profiles and the significance of partial/quarter LAH.
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Acknowledgements |
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Notes |
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References |
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Submitted on July 27, 2000; accepted on June 29, 2001.