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1 COMMENT THIS ARTICLE SEE COMMENTS ON THIS ARTICLE CONTACT AUTHOR 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 (p<0.01) in the implantation rate when compared to 24 untreated patients. Conclusion. The use of a 1.48 µm diode laser to drill an opening into the zona pellucida provides a good alternate to conventionally applied techniques. The procedure is efficient and safe as long as it is applied properly. In a human in vitro fertilization program, selected patients will have a benefit form assisted hatching. Key words: blastocyst; conception; embryo, preimplantation; lasers; fertilization in vitro; maternal age 35 and over; ovum-sperm interactions; reproduction; sperm penetration Up to the blastocyst stage, the mammalian embryo is surrounded by the zona pellucida. Prior to the implantation, the embryo has to escape from the zona pellucida, a process known as hatching. If this process is not completed properly, implantation will fail and a pregnancy cannot occur. It has been suspected that the artificial conditions during culture of human embryos in vitro may cause a hardening of the zona and thus constitute a primary reason for hatching difficulties (1-3). Other reasons for a possible impairment of the hatching process may be a thick zona pellucida (>20 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

2 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 (K /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

3 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<0.05) and versus the untreated control group (p<0.05). No statistical difference was found for the hatching rate of the untreated control group compared to the group with small openings. Table 1. The effect of openings in the zona pellucida with different size on the hatching process in the mouse Two Drilled Openings are Used for Hatching We investigated the effect of two openings (12-15 µm in size) which were laser-drilled at the zygote stage. Both openings were separated from each other by approximately one fourth of the circumference of the zona. The laser-drilling of two openings did not interfere with further development. Laser-drilled embryos started hatching 4 to 5 days after zygote recovery. Both laserdrilled openings were used for the escape through the zona pellucida (Fig. 2). None of these embryos was able to complete the hatching process. The embryos were trapped within the zona and finally degenerated. Figure 2. A mouse blastocyst at day 5. It was treated with the diode laser at the zygote stage to create two openings in the zona pellucida which were separated from each other by approximately one fourth of the circumference. Both openings were used for the escape from the zona and the blastocyst was trapped and did not complete hatching. Bar=10 µm. Figure 3. This expanded mouse blastocyst was laser-drilled at day 5. The drilled opening is denoted by an arrow (A). Within 5-10 min after laser-drilling the blastocyst collapsed (B, C) and started reexpansion after 2-4 hours (D). Subsequently, the blastocyst started hatching the following day (not shown). Bar=10 µm. Laser-Drilling at the Blastocyst Stage Retards Further Development We examined the effect of laser-drilling at the blastocyst stage, when the perivitelline space is no longer visible due to beginning of expansion of the trophectoderm which is in direct contact to the zona pellucida. Immediately after laser-drilling, blastocysts collapsed within 5 to 10 minutes and remained at that collapsed stage for another 1-2 hours. Reexpansion was observed 4 hours after

4 laser drilling and hatching started 12 to 16 hours later (Fig. 3). Embryos treated at an earlier stage of development were already hatched by that time. Laser-Assisted Hatching Proves Beneficial in Patients of Advanced Maternal Age Assisted hatching in the human required a higher laser doses due to the larger diameter of the human zona pellucida (Fig. 4). The benefit of assisted hatching was studied in a selected group of couples with advanced maternal age (over 35 years) receiving assisted reproduction treatment (in vitro fertilization and/or intracytoplasmic sperm injection). There was no significant difference between the control group and the treatment group in terms of age, previous treatment cycles, and mean number of embryos transferred in the actual treatment cycle. There was a slight trend towards a higher pregnancy rate in the group of patients who received assisted hatching (33.3% vs. 12.5%; p<0.1) although not yet statistically significant due to the small number of patients enrolled so far (Table 2). However, we found a significantly higher implantation rate per embryo transferred after laser treatment (22% with assisted hatching vs. 5.3% without assisted hatching; p<0.01). This resulted in 5 twin pregnancies in the assisted hatching group. All other pregnancies were singletons. Table 2. Assisted hatching in patients with advanced maternal age (mean±s.e.m.) Figure 4. A human embryo with 4 blastomeres before (A) and after (B) laser-dilling. Two laser shots at 18 ms pulse length were applied at the positions indicated by asterisks (A) and created the large opening (B). Immediately after laser-drilling the embryo was transferred back into the uterus. Bar=15 µm. (Reprinted by permission from Springer-Verlag GmbH & Co. KG from Montag M, Rink K, Delacrétaz G, van der Ven H. Die Anwendung der Lasertechnik im Bereich der assistierten Reproduktion; Reproduktionsmedizin 1999;15:45-54). Discussion Use of Lasers in Assisted Reproduction In this study we investigated some of the characteristic features of the application of a 1.48 µm diode laser for the creation of an opening in the zona pellucida. This laser was first introduced by Rink et al in 1994 (15). Although other types of laser were initially used for assisted fertilization (24), these lasers were difficult to use and were insufficiently cost-effective for routine application in assisted reproduction. It was the 1.48 µm diode laser which revolutionized the field of laser application in assisted reproduction (14,15). This laser allowed easy and efficient manipulation of the zona pellucida and its safety and efficacy was investigated in detail (17,22). Recently, numerous applications have been described, where the 1.48 µm Fertilase system was successfully used (25). We felt that some of the characteristic features for the application of this laser were not assessed and demonstrated in detail so far. The aim of our investigations was to show that this laser needs to be applied in the proper way in order to avoid problems which could interfere with subsequent hatching at the blastocyst stage. One such feature is the size of a drilled opening. For example, if the primary aim is the biopsy of the polar body, drilling of a rather small opening is sufficient for that purpose (18). However, our results clearly show that any opening present in the zona pellucida is used by the expanding blastocyst for hatching. If the opening is too small, a hatching blastocyst easily gets trapped. Therefore, hatching cannot be completed and consequently implantation will fail. A minimum size of an opening seems to be mandatory to allow efficient hatching. We found that in the mouse this size should be µm. As the diameter of the mouse zona pellucida during early embryogenesis is in the range of 6-7 µm, it may be concluded that doubling that value gives a good approximation for the size of an opening to allow for efficient and complete hatching. During laser-drilling, the introduction of more than one opening into the zona at different positions should be avoided. When we drilled two openings into the zona of mouse zygotes, both openings were used at the time of hatching. This, of course, leads to a perfect entrapment of the hatching blastocyst and consequently results in failed implantation. The use of the laser at the blastocyst stage needs to be carefully evaluated. Our experiments showed that as soon as laser-drilling is performed, blastocysts will collapse. At present, we cannot exclude that due to the close contact of the trophectodermal cells to the zona pellucida, the laser will hit the trophectoderm and cause a local damage. However, collapsed blastocysts reexpand within 4 hours and undergo hatching, although under these circumstances the time course of hatching is delayed by the laser treatment. This has to be taken into account if laser-drilling at the blastocyst stage needs to be performed, e.g., for blastocyst biopsy (26). Assisted Hatching The introduction of the 1.48 µm diode laser allowed for the first time to create very precise and

5 defined openings in the zona pellucida at a minimum requirement of time (15). Therefore this laser is excellently suited for assisted hatching (22). However, assisted hatching still is a controversial issue in assisted reproduction. Numerous studies have revealed no benefit of this technique (11-13, 27) while others reported significantly higher pregnancy rates (4,5,9,28-30). We believe that these controversial findings are due to the fact that most studies which were performed in the past employed either acidic Tyrode's solution to open the zona pellucida by chemical means (7) or used the mechanical way of partial zona dissection (8). Both procedures do not allow for the reliable production of equally sized holes. They require some experience and are time-consuming if compared to laser-drilling. Therefore, it is difficult to compare the results described for assisted hatching from different publications. Most studies do not even comment on the size of the drilled hole, although we know that the size of the drilled opening has a direct impact on the efficacy of blastocyst hatching in mouse (for laser-drilling this work; for zona drilling ref. 31, and in bovine model ref. 32). In order to assess the benefit of assisted hatching it is mandatory to create defined and precise openings into the zona pellucida. The use of lasers allows for the first time to meet this requirement (15,16). This may explain why we find a significant benefit in the implantation rate when we performed laserassisted hatching in patients with advanced maternal age compared to the most recent work by Lanzendorf et al (13) who reported on the contrary. However, these authors used acidic Tyrode's solution and it is possible that the openings they created did not meet the requirements of the blastocyst to hatch properly. Our results obtained with the mouse system clearly show that, in terms of hatching efficacy, the drilling of too small openings may be even worse than no treatment. Therefore we may conclude, that laser-assisted hatching can be beneficial, at least for selected patient groups. Although we focused on patients with advanced maternal age, an equally beneficial effect of assisted hatching may be seen if the technique is applied to patients with other indications. This may apply to patients with thick zona pellucida (4,33), elevated basal levels of follicle stimulating hormone (5,9) and recurrent implantation failure (29). These indications are under investigation in an ongoing multicentric study with the diode laser (23). Impact of Laser Technology on Assisted Reproduction The ease of application offered by the diode laser technology has stimulated a wide range of new developments in assisted reproduction. This includes all techniques which require an instantaneous and precise opening of the zona pellucida (18,21,26,34). Another new application is the use of this laser for controlled immobilization of spermatozoa and for the permeabilization of the sperm membrane (19,20,25). This technique may allow in the near future to perform the pretreatment of spermatozoa prior to intracytoplasmic sperm injection by the laser and without polyvinyl- pyrrolidone (PVP) which is discussed as a potentially harmful substance (35-38). The diode laser technology will definitely become of great importance for assisted reproduction. Acknowledgements We acknowledge the help of the IVF team at the University of Bonn and Mrs Przybylka for expert handling of our photographic demands. We are indebted to F. Schmoll (Institute for Animal Breeding at the University of Bonn) and to K. Rink (MTM Medical Technologies Montreux, Clarens, Montreux, Switzerland) for stimulating discussion. This research is an official part of the research performed at the University of Bonn. References 1 DeFelici M, Siracusa G. Spontaneous hardening of the zona pellucida of mouse oocytes during in vitro culture. Gamete Res 1982;6: Downs SM, Schroeder AC, Eppig JJ. Serum maintains the fertilizability of mouse oocytes matured in vitro by preventing hardening of the zona pellucida. Gamete Res 1986;15: Drobnis EZ, Andrew JB, Katz DF. Biophysical properties of the zona pellucida measured by suction: is zona hardening a mechanical phenomenon? Exp Zool 1988;245: Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, et al. Impairement of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 1990;5: Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992;7: Cohen J. Assisted hatching of human embryos. J In Vitro Fertil Embryo Transfer 1991;8: Gordon JW, Talansky BE. Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J Exp Zool 1987;239: Malter HE, Cohen J. Partial zona dissection of the human oocytes: a non-traumatic method using a

6 micromanipulation to assist zona pellucida penetration. Fertil Steril 1989;51: Schoolcraft WB, Schlenker T, Gee M, Jones GS, Jones HW. Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril 1994;62: Check JH, Hoover L, Nazari A, O'Shaughnessy A, Summers D. The effect of assisted hatching on pregnancy rates after frozen embryo transfer. Fertil Steril 1996;65: Hellebaut S, De Sutter P, Dozortsev D, Onghena A, Qian C, Dhont M. Does assisted hatching improve implantation rates after in vitro fertilization or intracytoplasmic sperm injection in all patients? A prospective randomized study. J Assist Reprod Genet 1996;13: Bider D, Livshits A, Yonish M, Yemini Z, Mashiach S, Dor J. Assisted hatching by zona drilling of human embryos in women of advanced age. Hum Reprod 1997;12: Lanzendorf SE, Nehchiri F, Mayer JF, Oehninger S, Muasher SJ. A prospective, randomized, double-blind study for the evaluation of assisted hatching in patients with advanced maternal age. Hum Reprod 1991;13: Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Aicardi G, Montanaro N. Rescue of implantation potential in embryos with poor prognosis by assisted hatching. Hum Reprod 1998;13: Rink K, Delacrétaz G, Salathé RP, Senn A, Nocera D, Germond M, et al mm diode laser microdissection of the zona pellucida of mouse zygotes. Proceedings SPIE 1994;213A: Rink K, Delacrétaz G, Salathé RP, Senn A, Nocera D, Germond M, et al. Non-contact microdrilling of mouse zona pellucida with an objective-delivered 1.48 mm diode laser. Lasers Surg Med 1996;18: Germond M, Nocera D, Senn A, Rink K, Delacrétaz G, Fakan S. Microdissection of mouse and human zona pellucida using a 1.48 mm diode laser beam: efficacy and safety of the procedure. Fertil Steril 1995;25: Montag M, van der Ven K, Delacrétaz G, Rink K, van der Ven H. Laser-assisted microdissection of zona pellucida facilitates polar body biopsy. Fertil Steril 1998a;69: Montag M, Rink K, Delacrétaz G, van der Ven H. Applications of a laser system for intracytoplasmic sperm injection. Abstracts of the 14th Annual Meeting of the European Society of Human Reproduction and Embryology; 1998 June 21-24; Göteborg, Sweden. Hum Reprod 1998(1);13: Montag M, Rink K, Delacrétaz G, van der Ven H. The potential use of a 1.48 µm diode lasersystem for immobilization and permeabilization of spermatozoa prior to intracytoplasmic sperm injection (ICSI). Abstracts of the 16th World congress on Fertility and Sterility; 1998 Oct 4-9; San Francisco (CA), USA. Amsterdam: Elsevier; Progr Suppl 1998c. p Montag M, Rink K, Dieckmann U, Delacrétaz G, van der Ven H. Laser-assisted cryopreservation of single human spermatozoa in cell-free zona pellucida. Andrologia 1999;32: Germond M, Senn A, Nocera D, Rink K, Delacrétaz G. Assisted hatching of frozen-thawed embryos with a 1.48 µm diode laser enhances pregnancy outcome in patients who had several previous nidation failures. Abstracts of the 54th Annual Meeting of the American Society for Reproductive Medicine; 1996 Nov 2-6; Boston (MA), USA. Amsterdam: Elsevier; Progr Suppl p Germond M, Primi MP, Senn A, Pannatier A, Rink K, Delacrétaz G, et al. Diode laser for assisted hatching: preliminary results of a multicentric prospective randomized study. Abstracts of the 14th Annual Meeting of the European Society of Human Reproduction and Embryology; 1998 June 21-24; Göteborg Sweden. Hum Reprod Hum Reprod 1998;13: Tadir Y. Ten years of laser-asisted gamete and embryo manipulation. Contemporary OB/GYN 1998;43: Montag M, Rink K, Descloux L, Delacrétaz G, van der Ven H. The use of a 1.48 µm laser-system in assisted reproduction: laser-drilling of the zona pellucida and laser-assisted immobilization of spermatozoa. Assisted Reprod. In press Veiga A, Sandalinas M, Benkhalifa M, Boada M, Carrera M, Santaló J, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 1997;5: Tucker MJ, Morton PC, Wright G Ingargiola DE, Sweitzer CL, Elsner CW, et al. Enhancement of outcome from intracytoplasmic sperm injection: does co-culture or assisted hatching improve implantation rates? Hum Reprod 1996;11: Schoolcraft WB, Schlenker T, Jones GS, Jones HW. In vitro fertilization in women age 40 and older: the impact of assisted hatching. J Assist Reprod Genet 1995;12: Stein A, Rufas O, Amit S, Avrech O, Pinkas H, Ovadia J, et al. Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in-vitro fertilization. Fertil Steril 1995;63: Chao KH, Chen SU, Chen HF, Wu MY, Yang YS, Ho HN. Assisted hatching increases the

7 implantation and pregnancy rate of in vitro fertilization (IVF)-embryo transfer (ET), but not that of IVFtubal ET in patients with repeated IVF failures. Fertil Steril 1997;67: Cohen J, Feldberg D. Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo. Mol Reprod Dev 1991;30: Schmoll F, Schneider H, Montag M, Rink K, Wimmers K, Tholen E, et al. Laser assisted hatching in bovine in vitro produced blastocysts. Theriogenology 1999;51: Cohen J, Inge KL, Suzman W, Wiker SR, Wright G. Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil Steril 1989;51: Boada M, Carrera M, De La Iglesia C, Sandalinas M, Barri PN, Veiga A. Successful use of a laser for human embryo biopsy in preimplantation genetic diagnosis: report of two cases. J Assist Reprod Genet 1997;15: Feichtinger W, Obruca A, Brunner M. Sex chromosomal abnormalities and intracytoplasmic injection. Lancet 1995;346: Jean M, Barriere P, Mirallie S. Intracytoplasmic sperm injection without polyvinylpyrrolidone: an essential precaution? Hum Reprod 1996;11: Butler EA, Mason, GM. Development of a successful ICSI Program without the use of PVP. Hum Reprod 1997;12: Strehler E, Baccetti B, Sterzik K, Capitani S, Collodel G, De Santo M, et al. Detrimental effects of polyvinyl- pyrrolidone on the ultrastructure of spermatozoa (Notulae seminologicae 13). Hum Reprod 1998;13: Received: April 7, 1999 Accepted: May 11, 1999 Correspondence to: Markus Montag Dept. of Endocrinology and Reproductive Medicine University of Bonn Sigmund-Freud-Str. 25 D Bonn, Germany m.montag@uni-bonn.de Copyright 1999 by the Croatian Medical Journal. All rights reserved. Created 22/7/99 - Last Modified 22/7/99 Created and maintained by: Tinman