The use of a 1.48 µm diode laser for assisted hatching was investigated in animal experimentation ... The safety of the system has been proven in animal studies ...
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March 1999 (Volume 40, Number 3)
Laser-Assisted Hatching in Assisted Reproduction Markus Montag, Hans van der Ven Department of Endocrinology and Reproductive Medicine, University of Bonn, Bonn, Germany Aim. The use of a 1.48 µm diode laser for assisted hatching was investigated in animal experimentation. Laser assisted hatching was offered to patients with advanced maternal age to evaluate a possible benefit. Methods. Using the Fertilase® system we investigated the impact of openings with different size in the zona of mouse embryos on the hatching process, as well as that of two openings. Laser-drilling was performed at the blastocyst stage to look for differences in timing and efficacy of hatching. The possible benefit of assisted hatching was studied in 24 couples with advanced maternal age (38.8±2.1 years) and compared to a control group (37.8±2.5 years) treated in the same time period but without assisted hatching. Results. A certain diameter of a laser drilled opening in the zona pellucida is necessary for efficient hatching. When two openings are present in the zona, the embryo will use both openings for hatching and subsequently become trapped. Laser-drilling at the expanded blastocyst stage causes an immediate collapse of treated blastocysts and the onset of hatching is retarded. Assisted hatching in 24 patients with advanced maternal age resulted in a significant increase (p20 µm) (4), elevated basal FSH (5), and advanced maternal age (6). In order to overcome hatching difficulties, Cohen et al proposed the creation of an artificial opening in the zona of an embryo prior to transfer (4), and this method became known as assisted hatching. The rational of this intervention clearly is to allow efficient embryo hatching through an artificial gap. Several methods have been employed for assisted hatching. Partial zona dissection by mechanical means using glass capillaries was widely used in the beginning (4). Later, chemical opening by means of acidic Tyrode's solution, also known as zona drilling (7), became the method of choice (5). Both methods require skill and are time-consuming and in particular the use of acidic Tyrode's solution has been debated as it may cause damage of the blastomeres (8). Therefore, the potential benefit of assisted hatching is still not fully accepted in the scientific community (9-14). The recent development of a non-contact 1.48 µm diode laser system offers an excellent method for zona manipulation (15,16). This laser is delivered through the objective and thus can be easily focused to allow for a touch-free laser application. The zona pellucida can be opened instantaneously with a single laser pulse of a few milliseconds duration. The safety of the system has been proven in animal studies (17). We have used the 1.48 µm diode laser for various applications in assisted reproduction, like polar body biopsy (18), immobilization of human spermatozoa and permeabilization of the sperm membrane (19,20), and cryopreservation of single spermatozoa in empty zona pellucida (21). The use of the Fertilase® system for assisted hatching has been shown already by Germond et al (22,23). In order to further elucidate its potential for assisted hatching, we examined certain characteristic features of the laser action on the zona pellucida from mouse embryos and blastocysts. We were
especially interested to see if the size and number of the drilled openings would have an impact on subsequent hatching. We further investigated the benefit of assisted hatching in a selected patient group at our in vitro fertilization (IVF) clinic, i.e., couples with advanced maternal age (over 35 years of age). Material and Methods Microscopic Setup and Laser Adaption We used a 1.48 µm diode laser system which was initially developed at the École Polytechnique Federale de Lausanne (15) and which is now available as a commercial system (Fertilase®, MTM Medical Technologies Montreux, Clarens, Montreux, Switzerland). The laser system was adapted to an inverted microscope (DMIRB, Leica, Bensheim, Germany) equipped with Hofman Modulation Contrast optics (Leica). The diode laser beam is guided through the optical system of the microscope and can be easily focused on the target. This wavelength does not require the use of special optics or culture dishes and therefore standard cell culture dishes can be employed. This characteristics allow for a touch-free delivery of the laser beam. Therefore, additional micromanipulation equipment is only necessary if further applications, like laser-assisted polar body or blastomere biopsy, are envisaged. Animal Experimentation According to federal law, we obtained permission to perform animal experimentation as presented in this study (K32 147-2737/3203). We used 12 week-old female CB6F1 and male NMRI mice. Females received an intraperitoneal injection of 5 I.U. of pregnant's mare serum gonadotropin, followed 46 hours later by 5 I.U. of human chorionic gondotropin (HCG). Females were mated and those presenting with vaginal plugs were used for the experiments. For the recovery of zygotes, mice were killed 20 hours after receiving HCG. Zygotes were flushed with phosphate buffered saline (PBS) out of the oviduct, denuded by hyaluronidase treatment (60 I.U. in Gamete-100 culture medium; Scandinavian IVF Sciences AB, Göteborg, Sweden), washed briefly in medium and incubated in 50 µL droplets of culture medium (IVF 50, IVF Sciences) under mineral oil (Ovoil, IVF Sciences). In a first experiment, we drilled openings of different size into the zona. For laser-drilling, culture dishes with the zygotes or 2-4 cell stage embryos were placed on the microscope stage. A tangential position of the zona pellucida of the embryos was focused and the laser treatment was applied by a single laser irradiation. The size of the drilled opening is dependent on the duration of the laser pulse. A longer pulse length corresponds to a higher energy doses and thus leads to a larger opening compared to a shorter pulse length. Mouse zonae were usually treated at a pulse length of 4 to 10 milliseconds (equivalent to an energy of 0.4 to 1 mJ at our setup). Following laser-drilling, embryos were washed twice in culture medium and were further incubated. The embryonic development was documented and special attention was paid to the onset of hatching as well as to the completion of the hatching process. Untreated zygotes served as a control. A second experiment was conducted as above, except that two or more openings were drilled within the zona from one embryo. Further evaluation was performed as described. In a third experiment we used embryos at the blastocyst stage when the perivitelline space was no longer visible and the trophectodermal cells were in direct contact to the zona pellucida (Fig. 1). Laser-drilling was performed using a pulse length of 6 ms. The effect of that laser application as well as the further development was assessed until the next day. Microphotography was performed using a MC-100 (Leica). Figure 1. A mouse blastocyst at day 7. It was laser-drilled at the zygote stage to create an opening of 5 µm. The blastocyst started hatching at day 4.5 and used the laser-drilled opening for the escape from the zona pellucida but subsequently got trapped and stopped further progression. Bar=10 µm. Assisted Hatching in Human In Vitro Fertilization We obtained permission from our institutional ethical review board to offer assisted hatching to selected patient groups. All patients receiving assisted hatching were informed and gave written consent. We investigated the possible benefit of assisted hatching in patients with advanced maternal age (over 35 years of age). Between June 1998 and February 1999, 24 patients aged over 35 years (38.8±2.1 years) received assisted hatching and these were all included in the evaluation presented here. Six patients received their first treatment cycle. The mean number of previous attempts for all patients was 2.3±1.3. In the actual treatment cycle, 8 patients were treated by conventional in vitro fertilization (IVF) and 16 by intracytoplasmic sperm injection (ICSI). The indications for IVF was mainly tubal factor and/or endocrinological problems, whereas ICSI was indicated in couples with male factor. The control group was matched retrospectively from patients who were treated in the same time period. The mean age for the control group was 37±2.5 years; 8 received a first treatment cycle
and for all patients in the control group the mean number of previous attempts was 2.2±1.5. Ten patients received IVF and 14 ICSI. Follicular stimulation was carried out by the combination of the gonadotrophin releasing hormone agonist (GnRHa) triptorelin actetate (Decapeptyl, Ferring, Kiel, Germany), human menopausal gonadotrophin (HMG; Humegon, Organon, Oberschleissheim, Germany) and/or recombinant follicular stimulating hormone (FSH; Gonal-F, Serono, Unterschleiss- heim, Germany) and human chorionic gonadotrophin (HCG). Triptorelin acetate (0.1 mg/day) was administered from day 22 of the previous cycle. Twelve to 15 days later, HMG/FSH was administered daily. Ovarian response was monitored by transvaginal ultrasound and HMG/FSH was adjusted according to the patient's response based on follicular size and estradiol levels. HCG (10,000 I.U.) was administered when the leading follicles were ³18 mm in diameter. Embryo transfer was performed at day two after oocyte retrieval at the 4-8 cell stage. In those patients where the embryos were treated by the laser, laser-assisted hatching was performed immediately before the transfer. An opening of 25 to 35 µm was drilled into the zona of human embryos by two neighboring laser shots using a pulse length of 18 ms (see Fig. 4).The thickness of the zona pellucida of embryos was measured at that time. An ongoing clinical pregnancy was defined as the presence of one or more gestational sacs by ultrasound at 6 weeks after transfer. For statistical evaluation the chi-square test was used. Results Efficacy of Laser-Assisted Hatching Depends on the Size of the Drilled Opening In a first experiment we determined the impact of the size of a laser-drilled opening on subsequent hatching. In one group of zygotes (48 zygotes) the size of the drilled openings was 5 µm and in another group (52 zygotes) 15 µm. Controls (50 zygotes) were not treated by the laser. The laser drilling did not interfere with further embryonic development and treated as well as untreated zygotes developed into blastocysts at comparable rates (81-84%). From those blastocysts which developed from laser-drilled zygotes with a larger opening, 90% were hatched by day 6. From those with smaller openings, 50% were hatched and in the untreated control group 69% hatched completely (Table 1). The hatching rate differed significantly between the group with large openings versus those with smaller openings (p
