TECHNIQUES AND INSTRUMENTATION

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1 FERTILITY AND STERILITY VOL. 80, NO. 5, NOVEMBER 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. TECHNIQUES AND INSTRUMENTATION Randomized controlled study of human zona pellucida dissection using the Zona Infrared Laser Optical System: evaluation of blastomere damage, embryo development, and subsequent Benjamin C. Wong, M.D., a Catherine A. Boyd, B.S., b and Susan E. Lanzendorf, Ph.D. c The Howard and Georgeanna Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia Received January 27, 2003; revised and accepted April 15, Supported by Hamilton- Thorne Biosciences Research, Beverly, Massachusetts. Presented at the 58th Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, October 12 17, Reprint requests: Benjamin C. Wong, M.D., The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia (FAX: ; E- mail: wongbc@evms.edu). a Department of Obstetrics and Gynecology, The Howard and Georgeanna Jones Institute for Reproductive Medicine. b OPELCO, Dulles, Virginia. c Washington University Medical School, St. Louis, Missouri /03/$30.00 doi: /s (03) Objective: To assess the effect of laser on human embryo damage and subsequent development using the Zona Infrared Laser Optical System (ZILOS). Design: Randomized controlled study. Setting: Tertiary care fertility clinic. Patient(s): One hundred fourteen donated and discarded frozen human embryos. Intervention(s): Embryos were thawed, cultured with cleavage and morphology evaluated periodically, and randomized into control, partial, or complete groups. The laser procedure was performed by ZILOS. Zona thickness and embryo diameter were recorded. Complete involved the production of a full-thickness defect in the zona and partial, a defect in the outer half of the zona. No laser treatment was administered to the control group. Main Outcome Measure(s): Blastocyst development and process. Result(s): No significant difference was noted between the three study groups for their baseline characteristics. There was no significant difference in blastocyst development among the three groups. However, the complete group showed a significant increase in compared to the control group. Conclusion(s): Complete laser of human embryos using the ZILOS does not have an adverse effect on subsequent development and increases the rate of. (Fertil Steril 2003;80: by American Society for Reproductive Medicine.) Key Words: Blastocyst, embryo cryopreservation, embryo culture, laser, ZILOS Assisted (AH) is a technique used by IVF programs in an effort to improve implantation and pregnancy rates. However, studies evaluating the implantation and pregnancy rates after AH have reported conflicting findings. Although some show an improvement in outcome (1 6), others show no benefit (7 10). The general suggestion is that patients whose embryos have a poor prognosis for implantation should consider AH. Various techniques have been used for AH, including chemical opening by acidified Tyrode s solution or protease, mechanical zona dissection, and laser beams. Assisted is typically performed by expelling an acidic solution through a fine micropipette over a small area to dissolve the zona. Mechanical partial zona dissection involves piercing or thinning the zona mechanically with a very thin glass microneedle. However, both of these techniques lack precision and reproducibility because they require the direct application of the pipettes and solutions by the human operator. The use of a laser for AH was developed to provide a more precise and controlled opening that can be standardized between specimens and operators (11). Two types of lasers have been evaluated for AH, the contact laser and the noncontact laser. The former requires some 1249

2 form of delivery assistance such as glass pipette or optical fiber to direct the laser beam against the zona, whereas the latter does not. Although pregnancies have been reported using the contact type of laser (12, 13), the need for specimen contact and specialized instruments has reduced the usefulness of this type for AH (14). On the other hand, the noncontact laser is technically easier to use and studies report that the laser light undergoes minimal absorption by the surrounding dish and medium and emits light at a safer wavelength (15). The Zona Infrared Laser Optical System (ZILOS, Hamilton-Thorne Research, Beverly, MA) used in our study utilizes a m infrared diode laser beam. The procedure is performed by directing the path of the laser beam through the objective on an inverted microscope and the precise position of the laser on the target is determined with the assistance of a computer. This allows the operator to control very precisely the size of the opening so that adjacent cells remain unaffected. A previous study investigated the thermal effects of its zona drilling on bovine and murine embryos (16). The use of a m infrared diode laser to perform AH is a widely accepted technique. However, in the United States, the Food and Drug Administration (FDA) considers the m infrared diode laser a class III device (highest risk device). IVF laboratories must first obtain an Investigational Device Exemption before using it on the embryos of IVF patients and many IVF programs need to obtain approval by the institutional review board to perform AH with a laser. The present randomized controlled study aims to assess the safety of laser AH using the ZILOS in human embryos and the subsequent of the embryos out of the zona pellucida in follow-up in vitro culture. This research study is sponsored by Hamilton-Thorne Research, Beverly, Massachusetts. Studies, like the one presented here, may enable the laser to be reclassified to a class II device and would not require IVF programs to obtain an Investigational Device Exemption before using the laser clinically. MATERIALS AND METHODS Human Embryos A total of 114 discarded cryopreserved human embryos donated for research from 34 consenting couples were used in this study following approval by the Institutional Review Board of the Eastern Virginia Medical School. Because the dates that the embryos were cryopreserved ranged from 1991 to 2000, it is difficult to describe the protocols for ovarian stimulation, insemination, embryo culture, and cryopreservation. In general, the protocols used were those previously described (17 24). Upon thaw, surviving embryos were cultured in sequential medium (Irvine Scientific, Santa Ana, CA) for 3 6 days depending on their cell stage at cryopreservation. Embryo cleavage and morphology were evaluated every 24 hours. Embryos were given a numerical grade at each evaluation using the following scale: grade 1 (blastomeres of equal size; no cytoplasmic fragments); grade 2 (blastomeres of equal size; minor cytoplasmic fragments or blebs); grade 3 (blastomeres of distinctly unequal size; few or no cytoplasmic fragments); grade 4 (blastomeres of equal or unequal size; significant cytoplasmic fragmentation); and grade 5 (few blastomeres of any size; severe or complete fragmentation). When embryos reached the blastocyst stage, they were graded as follows: grade 1 (completely expanded blastocoel cavity; inner cell mass well defined; no degeneration); grade 2 (completely expanded or very near; inner cell mass well defined; some areas of dark cells; some degenerated or live cells that are not incorporated within the blastocyst); and grade 3 (blastocoel cavity does not take up the entire zona; numerous cells or debris that is not a part of the blastocyst; dark cells within the blastocyst; inner cell mass may or may not be defined). To evaluate the effect of the laser procedure for embryo damage and subsequent development, the available day 2 3 frozen thawed embryos were randomized into three groups: control group, partial, and complete, according to the order in which they appeared on the patient information sheets. Embryos in the control group did not undergo laser AH, whereas the two test groups did. The embryos in all groups were evaluated and photographed before and after the laser procedure. Laser AH Procedure The laser procedure was performed on an Olympus IX-70 inverted microscope (Olympus America, Inc., Melville, NY) equipped with the ZILOS. Before performing the procedure, all embryos, including controls, were evaluated using the computer imager and the embryo diameter and zona thickness were recorded. Two measurements were made for the embryo diameter and five for zona thickness. The mean of these measurements was then calculated. To perform the procedure, each embryo was placed on the stage of the microscope and the laser aligned into the field. The embryo was positioned so that a portion of the zona was in the path of the laser beam. The laser beam was activated and the laser fired to create a hole in the zona. In the partial group, three pulses of laser at mw lasting 2 milliseconds each were administered to create a defect in the outer half of the zona pellucida (Fig. 1). In the complete group, two to four pulses of laser at the same power and duration were administered to ensure creation of a defect across the entire thickness of the zona pellucida (Fig. 2). The dimensions of the opening were recorded using the ZILOS computer scale and an image of the embryo recorded electronically for the output report. Two parameters were used to reflect the size of the opening. The diagonal distances between the opposite corners were 1250 Wong et al. Human embryo laser using the ZILOS Vol. 80, No. 5, November 2003

3 FIGURE 1 Photomicrograph of human eight-cell embryo after partial of the zona pellucida (arrows mark margins of defect). Original magnification, 400. recorded as drill and the width of the opening were recorded as ruler. Two measurements were performed for the former and three measurements for the latter. Means were obtained for these measurements. After, the embryo was returned to the incubator. Control embryos were also placed on the stage of the microscope but were only evaluated for embryo diameter and zona thickness. An image of each control embryo was also recorded. These embryos were returned to the incubator and cultured and monitored for development to the blastocyst stage and the process. After the initial part of the study, the partial group was dropped as a treatment arm in the randomization process as there appeared to be little difference between it and the control group. Due to the limited number of embryos available for the study, the change in protocol to randomization of the embryos equally between the control group and complete group allowed an increase in the power of the study to detect any differences in outcome between the two groups. Statistical analysis was performed using the Kruskal- Wallis test for the baseline parameters of different groups and the Fisher s exact test for the outcome measurements. Correlation between baseline characteristics and outcomes were also studied. Sample size calculation using an of 0.05 and a power of 0.8 revealed the need of 87 embryos to detect a 30% difference in the percentage of embryos reaching the blastocyst stage when the control group is compared with the complete group using a one-sided test. All embryos that underwent randomization were included in the analysis. RESULTS Baseline Characteristics of Embryos Randomized in the Study To determine whether the laser AH procedure resulted in observable damage to blastomeres or subsequent cleavage, a total of 114 embryos were evaluated (Table 1). There was no significant difference in the age of embryo donors at the time of oocyte retrieval, cell stage, and embryo grade at the time of freeze or thaw and embryo diameter among the three groups and between the control and the complete groups. Effect of Laser AH on Embryo Development Table 2 shows the overall different outcomes of each group as to the final stage reached by the embryos: embryos arresting before blastulation (arrested embryos), blastocyst development with no subsequent, and blastocyst development followed by subsequent. When all three groups are considered, there was no statistical difference. However, when the control group was compared with the complete group, there was a statistically significant difference with more blastocysts reaching the hatched stage in the complete group than in the control group. FIGURE 2 Photomicrograph of 10-cell human embryo after complete zona with the laser (arrows mark outer margins of the defect). Original magnification, 400. FERTILITY & STERILITY 1251

4 TABLE 1 Baseline characteristics of embryos. Group Control Complete Partial P Value n Age of donor at oocyte retrieval Cell stage at freeze Cell stage at thaw Cell stage at procedure Fragmentation (%) Embryo grade Zona thickness ( m) a Embryo size ( m) Ruler ( m) Drill ( m) a P.01 between control and complete groups; P.05 between control and partial groups; P.05 between complete and partial groups; Kruskal-Wallis test. TABLE 3 Outcome measurement: blastocyst development. Arrest before blastulation There was no significant difference in blastocyst development among the three groups or comparison between any of the two groups (Table 3). The mean grades of the blastocysts in the control, partial, and complete groups were 2.1, 2.0, and 2.0, respectively, which were not statistically different. This suggests that there was no evidence of blastomere damage to any of the embryos undergoing the laser procedure. The complete group showed a significant increase in the subsequent of the blastocyst out of the zona pellucida than the control group. The partial group did not show a significantly different rate of subsequent when compared to the control group or the complete group (Table 4). Correlation Between Baseline Characteristics and Successful Hatching of Embryos No statistically significant correlation was noted between any baseline characteristics with successful of embryos. More specifically, there was no statistically significant correlation between zona thickness and spontaneous in the three groups combined or within each individual group. However, there is a trend toward successful of the embryos with thicker zonae among the partial and the complete laser groups than in the control group. The zona thickness from the control, partial, and complete groups ranged from m, m, and m, respectively. Spontaneous occurred without any laser manipulation of zona for embryos with zona thickness ranging from m. Among the partial and complete laser groups, occurred for embryos of zona thickness from m and m, respectively. DISCUSSION Developed to blastocysts Control 25 (50%) 25 (50%) Complete 26 (50%) 26 (50%) Partial 4 (33%) 8 (67%) P.05 for comparison among all three groups and comparison between any two groups; Fisher exact test. One of the first noncontact laser systems used for AH was the 248-nm krypton fluoride excimer (25). However, there were theoretical mutagenic effects from this type of laser because the wavelength was close to the absorption peak of DNA at 260 nm. Therefore, subsequent studies in mice and human used lasers with less potential for DNA damage (26 30). The further introduction of an infrared diode laser emitting at 1.48 m allowed noncontact, microscope objective-delivered accessibility of laser light to the target with minimal absorption by the culture dish and the aqueous TABLE 2 Outcome measurement: overall embryo development. TABLE 4 Outcome measurement: blastocysts with subsequent successful. Arrest before blastulation Blastulation without Blastulation with Blastulation without Blastulation with Control 25 (50%) 23 (46%) 2 (4%) Complete 26 (50%) 16 (31%) 10 (19%) Partial 4 (33%) 6 (50%) 2 (17%) P.077 for all three groups; Fisher exact test. P.035 between control and complete groups; Fisher exact test. Control 23 (92%) 2 (8%) Complete 16 (62%) 10 (38%) Partial 6 (75%) 2 (25%) P.031 for all three groups; Fisher exact test. P.018 between control and complete groups; Fisher exact test Wong et al. Human embryo laser using the ZILOS Vol. 80, No. 5, November 2003

5 medium while the emitted wavelength is far from the absorption peak of DNA (15). A study comparing the photo stress effects of ultraviolet exposure and near infrared laser tweezers on human spermatozoa suggested that laser microbeam at the latter wavelength was safer (31). Data that support the use of the m diode laser for zona dissection are available. Significant increases in have been seen with its use in both the bovine (32) and mouse models (33) and laser-drilled mouse embryos have resulted in improved fertilization and implantation rates and normal, fertile offspring (34). These investigators saw no damage to the embryos during the laser procedure. A study using human embryos showed that laser zona thinning using a noncontact m diode led to significantly higher rates (35). Two other studies have evaluated its use for zona dissection for preimplantation genetic diagnosis (36, 37). Mantoudis et al. (38) performed laser AH on patients with the Fertilase micro drill (Fertilase Medical Technologies, Montreux SA, Switzerland), which is a m noncontact system, and noted an improved clinical pregnancy rate from embryos treated by quarter than partial or complete. There was no control group in the study. The dose of irradiation, which was one or two pulses of 47 mw each lasting 20 milliseconds, was higher than that used in our study. In comparison, each of these pulses only last 2 milliseconds in the ZILOS. The procedure is performed by directing the path of the laser beam through the objective of an inverted microscope and the precise position of the laser on the target is determined with the assistance of a computer. This allows the operator to control very precisely the size of the opening so that adjacent cells remain unaffected. A previous study investigating the thermal effects of its zona drilling on bovine and murine embryos (16) has established safety and efficacy data. The current study has established the safety and efficacy data in human embryos. In our initial phase, no benefit of partial laser over complete was seen. The m infrared diode laser used in the ZILOS for performing AH is a widely accepted technique. However, according to the Medical Device Amendments of 1976, devices that were not in commercial distribution at that time were called post-amendments devices and are classified automatically by statute (Section 513 (f)) into class III. Therefore, the majority of devices used for IVF and assisted reproductive technology (ART), such as ZILOS, defaulted into class III. In 1995, the FDA convened the Obstetrics and Gynecology Devices panel to classify as many IVF/ART devices that it knew about and were in commercial distribution at that time. Regarding lasers, the panel believed that there was insufficient information available to decide on their classification and they recommended that they remain in class III. The current statute provides for manufacturers or importers of a device to petition the FDA to reclassify the laser. The petition would need to have information such that it could render a decision on safety and effectiveness based on valid scientific evidence known to date. To date, there have been no submissions to the FDA for Pre-Market Approval or petitions to reclassify lasers for AH and they remain at a class III classification. For these reasons, basic and clinical research using m infrared diode lasers for AH should continue in the United States, although numerous studies may have been performed abroad. In this study, the absence of damage to blastomeres by the laser procedure is implied as there was no difference in the proportion of embryos developing to the blastocyst stage. There was also no increase in the degree of fragmentation among the blastomeres between the treatment and the control groups. We have included the baseline and outcome data of the partial group but the small number limits the power for a conclusion to be drawn. Although there is no difference in the percentage of embryos reaching blastocyst stage, no conclusion can be drawn on the safety of partial laser due to the lack of power. Embryo damage is unlikely to be a concern as safety has already been established in the complete laser group. After the initial phase of the randomized study using both of the two treatment arms and the control arm and no deleterious influence of the ZILOS procedure on the embryos was seen, the protocol of the study was altered with removal of the partial group from the randomization process. This allowed us to concentrate our study on comparing the full group with the control group, and optimizing the comparison of outcomes by reaching adequate power. There is a statistically significant increase in embryos undergoing subsequent in the full laser group compared with the control group, supporting the efficacy of the laser procedure. The embryos in all three groups were left in culture for up to 7 days and the proportion of them undergoing is comparable to other studies on culture of human embryos. Due to the small number of embryos randomized to the partial laser group, this study does not rule out the possibility of a statistically significant improvement in the of embryos following partial laser, had a larger number of embryos been recruited. Limitations of this study include the lack of information on assessment of various doses of the laser treatment and the establishment of ultimate clinical benefit of improvement of implantation and pregnancy rates. Due to the limited number of embryos in the study, we are also unable to establish the potential association between different embryo parameters such as zona thickness and the subsequent successful of the embryos. Although the upper limit of zona thickness is higher among embryos that underwent spontaneous in the complete laser group than in the control group, this difference may be due to chance alone. FERTILITY & STERILITY 1253

6 Now that the safety and enhancement of subsequent embryo after laser using the ZILOS have been established, further studies, including a prospective randomized clinical trial, should be performed to answer these questions. References 1. Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted using zona drilling of human embryos with poor prognosis. Hum Reprod 1992;7: Schoolcraft WB, Schlenker T, Gee M, Jones GS, Jones HW Jr. Assisted in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril 1994;62: Schoolcraft WB, Schlenker T, Jones GS, Jones HW Jr. In vitro fertilization in women age 40 and older: the impact of assisted. J Assist Reprod Genet 1995;12: Stein A, Rufas O, Amit S, Avrech O, Pinkas H, Ovadia J, et al. Assisted by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization. Fertil Steril 1995;63: Chao K-H, Chen SU, Chen HF, Wu MY, Yang YS, Ho HN. 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Zygote 1997;5: 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 1998;15: Mantoudis E, Podsiadly BT, Gorgy A, Venkat G, Craft IL. A comparison between quarter, partial and total laser assisted in selected infertility patients. Hum Reprod 2001;16: Wong et al. Human embryo laser using the ZILOS Vol. 80, No. 5, November 2003