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Comparison of laser-assisted hatching and acidified Tyrode’s hatching by evaluation of blastocyst development rates in sibling embryos: a prospective randomized trial
Comparison of laser-assisted hatching and acidified Tyrode’s hatching by evaluation of blastocyst development rates in sibling embryos: a prospective randomized trial Amy E. Jones, M.S., Graham Wright, B.S., Hilton I. Kort, M.D., Robert J. Straub, M.D., and Zsolt P. Nagy, M.D., Ph.D. Reproductive Biology Associates, Atlanta, Georgia
Objective: To assess two zona drilling methods in terms of blastocyst development rates using sister embryos. Design: Prospective, randomized study. Sister embryos of 14 patients were randomly assigned on day 3 to acidified Tyrode’s zona drilling or to laser zona drilling. After biopsy, subsequent embryo culture until the blastocyst stage (day 5) was performed. Setting: Private fertility center. Patient(s): Patients undergoing IVF–preimplantation genetic diagnosis. Intervention(s): Embryo biopsy using either laser-assisted hatching or acidified Tyrode’s hatching on sibling embryos and subsequent blastocyst development evaluation. Main Outcome Measure(s): Evaluation of blastocyst development in terms of degree of expansion and cell number in the inner cell mass and trophectoderm. Result(s): Blastocyst development rates (and blastocyst quality) were similarly high in both the acidified Tyrode’s hatching group and the laser-assisted hatching group. Conclusion(s): Laser hatching does not impair embryonic development to the blastocyst stage, demonstrating that laser-assisted hatching is a suitable alternative to the use of acidified Tyrode’s solution for zona drilling. (Fertil Steril威 2006;85:487–91. ©2006 by American Society for Reproductive Medicine.) Key Words: Laser-assisted hatching, acidified Tyrode’s hatching, preimplantation genetic diagnosis, blastocyst development
The blastocyst emerges from the zona pellucida (ZP) in a process known as hatching and begins implantation in the endometrial lining. The ZP thins, allowing the embryo to hatch. The biochemical mechanism of hatching has not been extensively studied but has been shown to be mediated by the embryo rather than externally (1, 2). Acid digestion of the ZP makes use of the fact that the ZP can be dissolved in a low-pH solution. Acidified Tyrode’s solution (AT solution; pH of 2.2 to 2.6) has been used for decades to remove the ZP in animal systems such as that of the mouse and was used clinically to assist fertilization (3) and to remove blastomeres for preimplantation genetic diagnosis (4). Typically, the embryo is held in place with a holding pipette while a glass micropipette with an internal diameter of 10 to 12 m is placed against the ZP and the AT solution is slowly expelled until a focal breach is observed (5). If an embryo has a cluster of extracellular fragments or has a local region in which the perivitelline space is comparatively expanded, the zona rent is created in this area to avoid potential damage to Received February 25, 2005; revised and accepted July 26, 2005. Reprint requests: Amy Jones, M.S., Nashville Fertility Center, 345 23rd Ave North Suite 401, Nashville, TN 37203 (FAX: 404-257-3314; E-mail: [email protected]).
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.07.1314
blastomeres adjacent to the site of acid digestion. The dimension of the hole created by acidified Tyrode’s hatching (ATH) is difficult to standardize because it is a function of the amount of solution deposited and the time required to create a breach (zona-specific resistance to dissolution) and because it is, in large measure, operator dependent. However, measurements of the hole produced by acid digestion indicate that with skilled operators, it is usually between 10 and 13 m near the inner surface and between 15 and 18 m at zona surface (6). Removal of a single blastomere for preimplantation genetic diagnosis (PGD) from day 3 embryo is a well established method. However, it is not known whether the method of choice for ZP drilling at the biopsy procedure would influence further embryonic development. Traditionally, in most PGD centers, perforation of the ZP is performed by using acidified Tyrode’s hatching (ATH), with only a few centers using laser assisted hatching (LAH), particularly in the United States. Because of the paucity of studies comparing outcomes of both methods after PGD, many centers are reluctant to switch to the simpler and faster laser drilling. Therefore, in the present study, we used sister embryos to compare the effect of the
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two alternative zona drilling methods on blastocyst developmental rates. MATERIALS AND METHODS Patient Population A total of 14 IVF-PGD cycles were enrolled in this study. Patients underwent the following stimulation regimens: controlled ovarian hyperstimulation using leuprolide acetate (TAP Pharmaceuticals, North Chicago, IL) down-regulation (n ⫽ 3), ganirelix acetate (Organon, West Orange, NJ; n ⫽ 6), or leuprolide acetate flare protocol (n ⫽ 5) with recombinant FSH (Gonal F; Serono, Norwell, MA or Follistim; Organon) and recombinant hCG (r-hCG; Ovidrel; Serono). Embryos from the same patients (eligible for biopsy: ⬎5 cells and ⬍30% fragmentation) were divided randomly into two groups on day 3. In group 1, embryos were submitted to acidified Tyrode’s hatching (ATH), and in group 2, embryos underwent laser-assisted hatching (LAH). Embryos were chosen for transfer by selecting the best-quality blastocysts, which were also considered to be normal after the use of 9-chromosome fluorescent in situ hybridization (FISH). Embryo transfer was performed with the Wallace Catheter (Irvine Scientific, San Diego, CA). In Vitro Fertilization and Embryo Culture After egg collection, oocytes were pooled in a 60 ⫻ 15 mm IVFTC Falcon dish (Becton, Dickinson Laboratories, Franklin Lakes, NJ) containing 5 mL of N-2-hydroxyethylpiperazine-N=2-ethanesulfonic acid (HEPES)– buffered modified human tubal fluid with 10% Serum Protein Substitute (SPS; Cooper/ Sage Surgical) overlaid with 5 mL of oil (Ovoil; Vitrolife, Vero Beach, FL). After removal of approximately 50% of the cumulus mass with 22-G needles, oocytes for conventional insemination were transferred to 40-L drops of IVF culture medium (Cooper/Sage Surgical) containing 10% SPS by volume. Culture drops were maintained under oil (Vitrolife) in 60-mm Nunc dishes (Nalge Nunclon International, Rochester, NY). Oocytes were inseminated in glucose-free fertilization medium (Cooper/Sage Surgical), and 18 –20 hours after conventional insemination or ICSI, resulting zygotes were transferred to glucose-containing cleavage medium (Cooper/Sage Surgical) that was supplemented with 15% SPS (Cooper/Sage Surgical). After the biopsy procedure, embryos were returned to cleavage medium culture until the morning of day 4 after egg collection, at which time embryos were transferred into 40-L drops of blastocyst culture medium (Cooper/Sage Surgical) supplemented with 15% SPS. Drops of Sage Blastocyst Medium culture were maintained under oil in 60-mm Nunc dishes (Nalge Nunclon International). Assisted Hatching and Embryo Biopsy Embryo biopsy was performed in 20-L drops of Ca2⫹Mg2⫹-free PBS (Sigma Chemical Corp, St. Louis, MO) maintained under oil (Ovoil; Vitrolife, Vero Beach, FL). 488
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Humagen microtools were used as follows: holding pipette, 150 m outer diameter; hatching needle; 12 m outer diameter; and embryo biopsy needle, 45 m outer diameter. To remove a blastomere, an opening was made in the zona by using AT solution (Cooper/Sage Surgical) or with a 1.48-m diode laser (Zilos-tk Laser, Hamilton Thorne Research, Beverly, MA). In both groups, an opening of the zona of 15–20 m was formed, followed by the removal of a single blastomere. The procedure was followed by washing the embryo through six 30-L drops of HEPES-buffered human tubal fluid (Cooper/Sage Surgical) under oil and then returning the embryo to cleavage medium (Cooper/Sage Surgical). Early Cleavage Stage Grading System On the day of embryo biopsy, early cleavage embryos were graded by using a modified version of the Puissant et al (7) system, according to cell number, fragmentation, uneven cells, and multinucleation; grade A represented the highest quality embryo, of seven to eight cells with little or no fragmentation, uneven cells, or multinucleation; grade D represented the poorest quality embryo, with fewer than five cells, ⬎30% fragmentation, and/or multinucleation. Blastocyst Grading System The blastocyst grading system used in this study was simply a modified version of the system employed by Gardner et al. (8). Degree of expansion and cell number in the inner cell mass and trophoblast were assessed to reach a final score of A, B, C, or D, with A, B, and C representing good-, medium-, or poor-quality blastocysts, respectively, and with grade D given to those embryos that were cleavage stage on days 5 and 6 after egg retrieval. Statistical Analysis Fisher’s exact test was applied. Statistical significance was established at the level of 5% (P⫽.05).
TABLE 1 Embryo biopsy details after zona drilling with acidified Tyrode’s hatching or laser-assisted hatching. Total no. Cycles Biopsied embryos Cells removed Intact blastomeres Lysed cells
ATH
LAH
14 59 61 59 (96.7) 2
14 62 66 62 (93.9) 4
Note: Data are n or n (%). Jones. Hatching methods and blastocyst development. Fertil Steril 2006.
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TABLE 2 Day 3 and 5 embryo quality in terms of cell stage and blastocyst development. Embryos by characteristics eligible for biopsy Embryos with ⬎5 cells on day 3 (n) Average cell stage on day 3 Blastocyst development, grades A and B combined (%)
ATH 59
LAH 62
7.37
7.09
47.5%
46.8%
Note: All P values comparing the two types of zona drilling were statistically nonsignificant. Jones. Hatching methods and blastocyst development. Fertil Steril 2006.
RESULTS The mean age (⫾SD) of the women in these 14 cycles were 36.9 (⫾3.9). A total of 285 oocytes were recovered, and 175 oocytes fertilized normally (61%). Seventy-two percent of the embryos underwent embryo biopsy, with an average of nine embryos biopsied per patient. Embryo biopsy details are summarized in Table 1. The time required to perform the embryo biopsy varied only slightly between the two methods of hatching, ranging between 30 and 90 seconds. Only when the zona was partially breached as a result of incomplete ATH did the procedure require up to 5 minutes to perform. Cell lysis occurred at a rate of 1.5% in the LAH group and of 3.2% in the ATH group. This difference was not significant and occurred during the actual biopsy process and apparently not as a result of the method of assisted hatching. An average of 1.06 cells and 1.03 cells were removed per embryo in the LAH group and ATH group, respectively. Data on the 14 cycles comparing AT zona drilling with laser hatching are presented in Tables 2 and 3. Early cleavage
stage embryo development was not statistically different between the two groups (Table 1). Using the early cleavage and blastocyst grading system described above, there was also no difference observed between the ATH and LAH embryos (Table 3). Seven patients achieved pregnancy (50%) after a total of 30 blastocysts (15 embryos from the ATH group and 15 embryos from the LAH group) were transferred. Patients having had transferred only ATH (n ⫽ 4) or only LAH (n ⫽ 5) embryos, or a combination of both (n ⫽ 5), had a pregnancy rate (defined by the presence of a fetal heart) of 50%, 60%, and 40%, respectively. DISCUSSION In the field of assisted reproductive technology, there has been a trend of highly invasive newly developed procedures that have been fundamental in our ability to provide care to patient populations with specific needs. The adoption of preimplantation genetic diagnosis into an IVF lab daily routine, although enormously valuable, can be a daunting task in terms of staff training, education, and scheduling. Thus, with the development of new technologies that allow for more rapid execution of a methodology, an expedient shift in protocol implementation often is preferred. However, thorough evaluation of newly introduced techniques should be performed to confirm the safety and efficacy of the procedure. Here, in an effort to assess the effect of the diode laser on blastocyst development after embryo biopsy, we evaluated embryo morphology in two groups of randomly assigned sister embryos that had undergone LAH or acid digestion of the ZP. Assisted hatching is defined as an artificial breaching of the ZP and was first introduced in clinical IVF by Malter and Cohen (9) and Cohen et al. (10) as an artificial means for the embryo to escape to improve clinical pregnancy rates. This technique is still being applied as a method to reduce the effect of putative iatrogenic ZP hardening (11). Additionally, the method of zona penetration has made it possible to access the contents of the oocyte or embryo for procedures such as
TABLE 3 Day 3 and 5 embryo quality in terms of grades assigned to the particular day of development. Embryo quality by stage Day 3 stage ATH embryos LAH embryos Day 5–6 stage ATH embryos LAH embryos
Grade A
Grade B
Grade C
Grade D
24 (40.7) 35 (56.4)
26 (44.1) 16 (25.8)
8 (13.6) 10 (16.1)
1 (1.7) 1 (1.6)
2 (3.4) 2 (3.2)
26 (44.1) 27 (43.5)
14 (23.7) 18 (29.0)
17 (28.8) 15 (24.2)
Note: All data are n (%). Jones. Hatching methods and blastocyst development. Fertil Steril 2006.
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PGD, extracellular fragment removal (12), and lysed cell removal (13). Although a learning curve is expected when this technique is used, only experienced embryologists participated in this study. Extreme care was taken to ensure that embryos were in the Ca2⫹Mg2⫹-free PBS for a minimal amount of time, and the use of a dual tool holder for the ATH cases minimized the time necessary for the procedure. Several techniques have been used to breach the zona, including mechanical hatching (14), acid digestion or zona drilling (5), LAH (15), and enzymatic digestion of the ZP (16). A recent retrospective study comparing these techniques for assisted hatching showed no difference in implantation or clinical pregnancy rate in the four groups (17). The most commonly used methods of assisted hatching in the clinical PGD setting are partial zona dissection, LAH, and zona drilling using acidified medium. Here, we compared the effect of the LAH and zona drilling on blastocyst developmental rates (using sister embryos). Laser-assisted hatching or photoablation of the ZP has been used to enhance fertilization in certain cases of male-factor infertility (18), to improve pregnancy rates in repeated implantation failures after IVF (19), and to facilitate early cleavage embryo and blastocyst biopsy for PGD (20, 21). The ideal laser for assisted hatching provides photoablation with light at a wavelength not absorbed by DNA and that will not produce heat. Typically, a system that conducts light to the ZP via fiber optic is attached to a micromanipulator (15) and requires manipulation very similar to that used in acid drilling. A number of different laser systems that use different sources that offer different wavelengths and therefore unique energetic properties have been proposed for clinical AH. The advantage of this system is that no micromanipulator is required, and adjustment of the microscope stage moves the embryo and zona into the path of the laser (22, 23). The laser creates a tangential furrow through the ZP, whose diameter is determined by focus of the laser beam and pulse duration. Complete rupture of the ZP often requires multiple firings of the laser, with more of the zona moved into the laser path each time. Standardization of the rent diameter is one clear advantage of this method over acid or enzymatic drilling, which makes this an appealing technique to integrate into procedures such as PGD. Recently, Joris et al. (24) retrospectively compared pregnancy outcomes in patients undergoing PGD using AT medium or a laser, and the results showed similar pregnancy rates in both groups. Laser-assisted hatching also is a comparatively easy technique that involves a short learning curve, requires a short amount of time to perform, and is extremely precise. It also can be incorporated into other procedures such as ICSI to improve outcomes in difficult cases (25). Concerns regarding thermal damage as a result of the proximity of the laser beam to the blastomeres, particularly in PGD cases in which hatching is performed immediately adjacent to the cell, appear to be unwarranted. Despite reported embryo 490
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survival rates following embryo biopsy (24, 26), we believe that a fundamental method of evaluating one’s embryo biopsy technique is to assess embryo developmental potential in extended culture. While embryo intactness may be a crude indication of viability it is not an indication of quality. Blastocyst development rates (and blastocyst quality) were similarly high in both the chemical zona drilling group and in the LAH group, indicating that laser hatching does not impair embryonic development to the blastocyst stage. Implantation rates were not compared as patients often had a combination of ATH and LAH embryos used for transfer. However, the high proportion of laser-drilled embryos transferred, together with the high pregnancy rate, is suggestive that zona drilling by laser microbeam does not produce additional risks for embryonic development beyond the blastocyst stage. Therefore, LAH can be recommended as an alternate technique that can replace the more laborious methods of breaching the ZP. REFERENCES 1. Perona RM, Wassarman PM. Mouse blastocysts hatch in vitro by using a trypsin-like proteinase associated with cells of mural trophectoderm. Dev Biol 1986;114:42–52. 2. Mishra A, Seshagiri PB. Evidence for the involvement of a speciesspecific embryonic protease in zona escape of hamster blastocysts. Mol Hum Reprod 2000;6:1005–12. 3. Gordon JW, Talansky BE. Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J Exp Zool 1986;239:347–54. 4. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific amplification. Nature 1990;344:378 – 80. 5. Cohen J, Malter HE, Talansky BE, Grifo J, eds. Micromanipulation of human oocytes and embryos. New York: Raven Press, 1991:191–222. 6. Tao J, Tamis RJ. Application of assisted hatching for 2-day-old, frozenthawed embryo transfer in a poor-prognosis population. Assist Reprod Genet 1997;14:128 –30. 7. Puissant F, Van Rysselberge M, Barlow P, Deweze J, Leroy F. Embryo scoring as a prognostic tool in IVF treatment. Hum Reprod 1987;2: 705– 8. 8. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73:1155– 8. 9. Malter HE, Cohen J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res 1989; 24:67– 80. 10. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 1990;5:7–13. 11. Tucker M, Cohen J, Massey J, Mayer M, Wiker S, Wright G. Partial zona dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am J Obstet Gynecol 1991;165:341– 4. 12. Alikani M, Cohen J, Tomkin G, Garrisi J, Mack C, Scott R. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril 1999;71:836 – 42. 13. Rienzi L, Nagy ZP, Ubaldi F, Iacobelli M, Anniballo R, Tesarik J, et al. Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged. Fertil Steril 2002;77:1196 – 201. 14. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP. Immunosuppresion supports implantation of zona pellucida dissected human embryos. Fertil Steril 1990;53:662–5.
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15. Feichtinger W, Strohmer H, Fuhrberg P, Radivojevic K, Antinori S, Pepe G, et al. Photablation of oocyte zona pellucida by erbium-YAG laser for in-vitro fertilisation in severe male infertility. Lancet 1992;339:811. 16. Fong CH, Bongso A, Ng AC, Anandakumar C, Trounson A, Ratnam S. Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in vitro fertilization and understanding implantation. Hum Reprod 1998;13:2926 –32. 17. Balaban B, Urman B, Alatas C, Mercan R, Mumcu A, Isiklar A. A comparison of four different techniques of assisted hatching. Hum Reprod 2002;17:1239 – 43. 18. Obruca A, Strohmer H, Sakkas D, Menezo Y, Kogosowski A, Barak Y, et al. Use of lasers in assisted fertilization and hatching. Hum Reprod 1994;9:1723– 6. 19. Mantoudis E, Podsiadly BT, Gorgy A, Venkat G, Craft IL. A comparison between quarter, partial and total laser assisted hatching in selected infertility patients. Hum Reprod 2001;16:2182– 6. 20. Bouda 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;15:302–7.
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21. Veiga A, Sandalinas M, Benkhalifa M, Boada M, Carrera M, Santalo J, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 1997;5:351– 4. 22. Neev J, Gonzales A, Licciardi F, Alikani M, Tadir Y, Berns M, et al. Opening of the mouse zona pellucida by laser without a micromanipulator. Hum Reprod 1993;8:939 – 44. 23. Neev J, Schiewe MC, Sung VW, Kang D, Hezeleger N, Berns MW, et al. Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system. J Assist Reprod Genet 1995;12:288 –93. 24. Joris H, De Vos A, Janssens R, Devroey P, Liebaers I, Van Steirteghem A. Comparison of the results of human embryo biopsy and outcome of PGD after zona drilling using acid Tyrode medium or a laser. Hum Reprod 2003;18:1896 –902. 25. Nagy ZP, Oliveira SA, Abdelmassih V, Abdelmassih R. Novel use of laser to assist ICSI for patients with fragile oocytes: a case report. Reprod Biomed Online 2002;4:27–31. 26. Harper J, Thornhill A. In: Harper J, Delhanty J, Handyside A, eds. Embryo biopsy in preimplantation genetic diagnosis. John Wiley and Sons, 2001:142– 63.
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Objective: To assess two zona drilling methods in terms of blastocyst development rates using sister embryos. Design: Prospective, randomized study. Sister embryos of 14 patients were randomly assigned on day 3 to acidified Tyrode’s zona drilling or to laser zona drilling. After biopsy, subsequent embryo culture until the blastocyst stage (day 5) was performed. Setting: Private fertility center. Patient(s): Patients undergoing IVF–preimplantation genetic diagnosis. Intervention(s): Embryo biopsy using either laser-assisted hatching or acidified Tyrode’s hatching on sibling embryos and subsequent blastocyst development evaluation. Main Outcome Measure(s): Evaluation of blastocyst development in terms of degree of expansion and cell number in the inner cell mass and trophectoderm. Result(s): Blastocyst development rates (and blastocyst quality) were similarly high in both the acidified Tyrode’s hatching group and the laser-assisted hatching group. Conclusion(s): Laser hatching does not impair embryonic development to the blastocyst stage, demonstrating that laser-assisted hatching is a suitable alternative to the use of acidified Tyrode’s solution for zona drilling. (Fertil Steril威 2006;85:487–91. ©2006 by American Society for Reproductive Medicine.) Key Words: Laser-assisted hatching, acidified Tyrode’s hatching, preimplantation genetic diagnosis, blastocyst development
The blastocyst emerges from the zona pellucida (ZP) in a process known as hatching and begins implantation in the endometrial lining. The ZP thins, allowing the embryo to hatch. The biochemical mechanism of hatching has not been extensively studied but has been shown to be mediated by the embryo rather than externally (1, 2). Acid digestion of the ZP makes use of the fact that the ZP can be dissolved in a low-pH solution. Acidified Tyrode’s solution (AT solution; pH of 2.2 to 2.6) has been used for decades to remove the ZP in animal systems such as that of the mouse and was used clinically to assist fertilization (3) and to remove blastomeres for preimplantation genetic diagnosis (4). Typically, the embryo is held in place with a holding pipette while a glass micropipette with an internal diameter of 10 to 12 m is placed against the ZP and the AT solution is slowly expelled until a focal breach is observed (5). If an embryo has a cluster of extracellular fragments or has a local region in which the perivitelline space is comparatively expanded, the zona rent is created in this area to avoid potential damage to Received February 25, 2005; revised and accepted July 26, 2005. Reprint requests: Amy Jones, M.S., Nashville Fertility Center, 345 23rd Ave North Suite 401, Nashville, TN 37203 (FAX: 404-257-3314; E-mail: [email protected]).
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.07.1314
blastomeres adjacent to the site of acid digestion. The dimension of the hole created by acidified Tyrode’s hatching (ATH) is difficult to standardize because it is a function of the amount of solution deposited and the time required to create a breach (zona-specific resistance to dissolution) and because it is, in large measure, operator dependent. However, measurements of the hole produced by acid digestion indicate that with skilled operators, it is usually between 10 and 13 m near the inner surface and between 15 and 18 m at zona surface (6). Removal of a single blastomere for preimplantation genetic diagnosis (PGD) from day 3 embryo is a well established method. However, it is not known whether the method of choice for ZP drilling at the biopsy procedure would influence further embryonic development. Traditionally, in most PGD centers, perforation of the ZP is performed by using acidified Tyrode’s hatching (ATH), with only a few centers using laser assisted hatching (LAH), particularly in the United States. Because of the paucity of studies comparing outcomes of both methods after PGD, many centers are reluctant to switch to the simpler and faster laser drilling. Therefore, in the present study, we used sister embryos to compare the effect of the
Fertility and Sterility姞 Vol. 85, No. 2, February 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
487
two alternative zona drilling methods on blastocyst developmental rates. MATERIALS AND METHODS Patient Population A total of 14 IVF-PGD cycles were enrolled in this study. Patients underwent the following stimulation regimens: controlled ovarian hyperstimulation using leuprolide acetate (TAP Pharmaceuticals, North Chicago, IL) down-regulation (n ⫽ 3), ganirelix acetate (Organon, West Orange, NJ; n ⫽ 6), or leuprolide acetate flare protocol (n ⫽ 5) with recombinant FSH (Gonal F; Serono, Norwell, MA or Follistim; Organon) and recombinant hCG (r-hCG; Ovidrel; Serono). Embryos from the same patients (eligible for biopsy: ⬎5 cells and ⬍30% fragmentation) were divided randomly into two groups on day 3. In group 1, embryos were submitted to acidified Tyrode’s hatching (ATH), and in group 2, embryos underwent laser-assisted hatching (LAH). Embryos were chosen for transfer by selecting the best-quality blastocysts, which were also considered to be normal after the use of 9-chromosome fluorescent in situ hybridization (FISH). Embryo transfer was performed with the Wallace Catheter (Irvine Scientific, San Diego, CA). In Vitro Fertilization and Embryo Culture After egg collection, oocytes were pooled in a 60 ⫻ 15 mm IVFTC Falcon dish (Becton, Dickinson Laboratories, Franklin Lakes, NJ) containing 5 mL of N-2-hydroxyethylpiperazine-N=2-ethanesulfonic acid (HEPES)– buffered modified human tubal fluid with 10% Serum Protein Substitute (SPS; Cooper/ Sage Surgical) overlaid with 5 mL of oil (Ovoil; Vitrolife, Vero Beach, FL). After removal of approximately 50% of the cumulus mass with 22-G needles, oocytes for conventional insemination were transferred to 40-L drops of IVF culture medium (Cooper/Sage Surgical) containing 10% SPS by volume. Culture drops were maintained under oil (Vitrolife) in 60-mm Nunc dishes (Nalge Nunclon International, Rochester, NY). Oocytes were inseminated in glucose-free fertilization medium (Cooper/Sage Surgical), and 18 –20 hours after conventional insemination or ICSI, resulting zygotes were transferred to glucose-containing cleavage medium (Cooper/Sage Surgical) that was supplemented with 15% SPS (Cooper/Sage Surgical). After the biopsy procedure, embryos were returned to cleavage medium culture until the morning of day 4 after egg collection, at which time embryos were transferred into 40-L drops of blastocyst culture medium (Cooper/Sage Surgical) supplemented with 15% SPS. Drops of Sage Blastocyst Medium culture were maintained under oil in 60-mm Nunc dishes (Nalge Nunclon International). Assisted Hatching and Embryo Biopsy Embryo biopsy was performed in 20-L drops of Ca2⫹Mg2⫹-free PBS (Sigma Chemical Corp, St. Louis, MO) maintained under oil (Ovoil; Vitrolife, Vero Beach, FL). 488
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Humagen microtools were used as follows: holding pipette, 150 m outer diameter; hatching needle; 12 m outer diameter; and embryo biopsy needle, 45 m outer diameter. To remove a blastomere, an opening was made in the zona by using AT solution (Cooper/Sage Surgical) or with a 1.48-m diode laser (Zilos-tk Laser, Hamilton Thorne Research, Beverly, MA). In both groups, an opening of the zona of 15–20 m was formed, followed by the removal of a single blastomere. The procedure was followed by washing the embryo through six 30-L drops of HEPES-buffered human tubal fluid (Cooper/Sage Surgical) under oil and then returning the embryo to cleavage medium (Cooper/Sage Surgical). Early Cleavage Stage Grading System On the day of embryo biopsy, early cleavage embryos were graded by using a modified version of the Puissant et al (7) system, according to cell number, fragmentation, uneven cells, and multinucleation; grade A represented the highest quality embryo, of seven to eight cells with little or no fragmentation, uneven cells, or multinucleation; grade D represented the poorest quality embryo, with fewer than five cells, ⬎30% fragmentation, and/or multinucleation. Blastocyst Grading System The blastocyst grading system used in this study was simply a modified version of the system employed by Gardner et al. (8). Degree of expansion and cell number in the inner cell mass and trophoblast were assessed to reach a final score of A, B, C, or D, with A, B, and C representing good-, medium-, or poor-quality blastocysts, respectively, and with grade D given to those embryos that were cleavage stage on days 5 and 6 after egg retrieval. Statistical Analysis Fisher’s exact test was applied. Statistical significance was established at the level of 5% (P⫽.05).
TABLE 1 Embryo biopsy details after zona drilling with acidified Tyrode’s hatching or laser-assisted hatching. Total no. Cycles Biopsied embryos Cells removed Intact blastomeres Lysed cells
ATH
LAH
14 59 61 59 (96.7) 2
14 62 66 62 (93.9) 4
Note: Data are n or n (%). Jones. Hatching methods and blastocyst development. Fertil Steril 2006.
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TABLE 2 Day 3 and 5 embryo quality in terms of cell stage and blastocyst development. Embryos by characteristics eligible for biopsy Embryos with ⬎5 cells on day 3 (n) Average cell stage on day 3 Blastocyst development, grades A and B combined (%)
ATH 59
LAH 62
7.37
7.09
47.5%
46.8%
Note: All P values comparing the two types of zona drilling were statistically nonsignificant. Jones. Hatching methods and blastocyst development. Fertil Steril 2006.
RESULTS The mean age (⫾SD) of the women in these 14 cycles were 36.9 (⫾3.9). A total of 285 oocytes were recovered, and 175 oocytes fertilized normally (61%). Seventy-two percent of the embryos underwent embryo biopsy, with an average of nine embryos biopsied per patient. Embryo biopsy details are summarized in Table 1. The time required to perform the embryo biopsy varied only slightly between the two methods of hatching, ranging between 30 and 90 seconds. Only when the zona was partially breached as a result of incomplete ATH did the procedure require up to 5 minutes to perform. Cell lysis occurred at a rate of 1.5% in the LAH group and of 3.2% in the ATH group. This difference was not significant and occurred during the actual biopsy process and apparently not as a result of the method of assisted hatching. An average of 1.06 cells and 1.03 cells were removed per embryo in the LAH group and ATH group, respectively. Data on the 14 cycles comparing AT zona drilling with laser hatching are presented in Tables 2 and 3. Early cleavage
stage embryo development was not statistically different between the two groups (Table 1). Using the early cleavage and blastocyst grading system described above, there was also no difference observed between the ATH and LAH embryos (Table 3). Seven patients achieved pregnancy (50%) after a total of 30 blastocysts (15 embryos from the ATH group and 15 embryos from the LAH group) were transferred. Patients having had transferred only ATH (n ⫽ 4) or only LAH (n ⫽ 5) embryos, or a combination of both (n ⫽ 5), had a pregnancy rate (defined by the presence of a fetal heart) of 50%, 60%, and 40%, respectively. DISCUSSION In the field of assisted reproductive technology, there has been a trend of highly invasive newly developed procedures that have been fundamental in our ability to provide care to patient populations with specific needs. The adoption of preimplantation genetic diagnosis into an IVF lab daily routine, although enormously valuable, can be a daunting task in terms of staff training, education, and scheduling. Thus, with the development of new technologies that allow for more rapid execution of a methodology, an expedient shift in protocol implementation often is preferred. However, thorough evaluation of newly introduced techniques should be performed to confirm the safety and efficacy of the procedure. Here, in an effort to assess the effect of the diode laser on blastocyst development after embryo biopsy, we evaluated embryo morphology in two groups of randomly assigned sister embryos that had undergone LAH or acid digestion of the ZP. Assisted hatching is defined as an artificial breaching of the ZP and was first introduced in clinical IVF by Malter and Cohen (9) and Cohen et al. (10) as an artificial means for the embryo to escape to improve clinical pregnancy rates. This technique is still being applied as a method to reduce the effect of putative iatrogenic ZP hardening (11). Additionally, the method of zona penetration has made it possible to access the contents of the oocyte or embryo for procedures such as
TABLE 3 Day 3 and 5 embryo quality in terms of grades assigned to the particular day of development. Embryo quality by stage Day 3 stage ATH embryos LAH embryos Day 5–6 stage ATH embryos LAH embryos
Grade A
Grade B
Grade C
Grade D
24 (40.7) 35 (56.4)
26 (44.1) 16 (25.8)
8 (13.6) 10 (16.1)
1 (1.7) 1 (1.6)
2 (3.4) 2 (3.2)
26 (44.1) 27 (43.5)
14 (23.7) 18 (29.0)
17 (28.8) 15 (24.2)
Note: All data are n (%). Jones. Hatching methods and blastocyst development. Fertil Steril 2006.
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PGD, extracellular fragment removal (12), and lysed cell removal (13). Although a learning curve is expected when this technique is used, only experienced embryologists participated in this study. Extreme care was taken to ensure that embryos were in the Ca2⫹Mg2⫹-free PBS for a minimal amount of time, and the use of a dual tool holder for the ATH cases minimized the time necessary for the procedure. Several techniques have been used to breach the zona, including mechanical hatching (14), acid digestion or zona drilling (5), LAH (15), and enzymatic digestion of the ZP (16). A recent retrospective study comparing these techniques for assisted hatching showed no difference in implantation or clinical pregnancy rate in the four groups (17). The most commonly used methods of assisted hatching in the clinical PGD setting are partial zona dissection, LAH, and zona drilling using acidified medium. Here, we compared the effect of the LAH and zona drilling on blastocyst developmental rates (using sister embryos). Laser-assisted hatching or photoablation of the ZP has been used to enhance fertilization in certain cases of male-factor infertility (18), to improve pregnancy rates in repeated implantation failures after IVF (19), and to facilitate early cleavage embryo and blastocyst biopsy for PGD (20, 21). The ideal laser for assisted hatching provides photoablation with light at a wavelength not absorbed by DNA and that will not produce heat. Typically, a system that conducts light to the ZP via fiber optic is attached to a micromanipulator (15) and requires manipulation very similar to that used in acid drilling. A number of different laser systems that use different sources that offer different wavelengths and therefore unique energetic properties have been proposed for clinical AH. The advantage of this system is that no micromanipulator is required, and adjustment of the microscope stage moves the embryo and zona into the path of the laser (22, 23). The laser creates a tangential furrow through the ZP, whose diameter is determined by focus of the laser beam and pulse duration. Complete rupture of the ZP often requires multiple firings of the laser, with more of the zona moved into the laser path each time. Standardization of the rent diameter is one clear advantage of this method over acid or enzymatic drilling, which makes this an appealing technique to integrate into procedures such as PGD. Recently, Joris et al. (24) retrospectively compared pregnancy outcomes in patients undergoing PGD using AT medium or a laser, and the results showed similar pregnancy rates in both groups. Laser-assisted hatching also is a comparatively easy technique that involves a short learning curve, requires a short amount of time to perform, and is extremely precise. It also can be incorporated into other procedures such as ICSI to improve outcomes in difficult cases (25). Concerns regarding thermal damage as a result of the proximity of the laser beam to the blastomeres, particularly in PGD cases in which hatching is performed immediately adjacent to the cell, appear to be unwarranted. Despite reported embryo 490
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survival rates following embryo biopsy (24, 26), we believe that a fundamental method of evaluating one’s embryo biopsy technique is to assess embryo developmental potential in extended culture. While embryo intactness may be a crude indication of viability it is not an indication of quality. Blastocyst development rates (and blastocyst quality) were similarly high in both the chemical zona drilling group and in the LAH group, indicating that laser hatching does not impair embryonic development to the blastocyst stage. Implantation rates were not compared as patients often had a combination of ATH and LAH embryos used for transfer. However, the high proportion of laser-drilled embryos transferred, together with the high pregnancy rate, is suggestive that zona drilling by laser microbeam does not produce additional risks for embryonic development beyond the blastocyst stage. Therefore, LAH can be recommended as an alternate technique that can replace the more laborious methods of breaching the ZP. REFERENCES 1. Perona RM, Wassarman PM. Mouse blastocysts hatch in vitro by using a trypsin-like proteinase associated with cells of mural trophectoderm. Dev Biol 1986;114:42–52. 2. Mishra A, Seshagiri PB. Evidence for the involvement of a speciesspecific embryonic protease in zona escape of hamster blastocysts. Mol Hum Reprod 2000;6:1005–12. 3. Gordon JW, Talansky BE. Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J Exp Zool 1986;239:347–54. 4. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific amplification. Nature 1990;344:378 – 80. 5. Cohen J, Malter HE, Talansky BE, Grifo J, eds. Micromanipulation of human oocytes and embryos. New York: Raven Press, 1991:191–222. 6. Tao J, Tamis RJ. Application of assisted hatching for 2-day-old, frozenthawed embryo transfer in a poor-prognosis population. Assist Reprod Genet 1997;14:128 –30. 7. Puissant F, Van Rysselberge M, Barlow P, Deweze J, Leroy F. Embryo scoring as a prognostic tool in IVF treatment. Hum Reprod 1987;2: 705– 8. 8. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73:1155– 8. 9. Malter HE, Cohen J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res 1989; 24:67– 80. 10. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 1990;5:7–13. 11. Tucker M, Cohen J, Massey J, Mayer M, Wiker S, Wright G. Partial zona dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am J Obstet Gynecol 1991;165:341– 4. 12. Alikani M, Cohen J, Tomkin G, Garrisi J, Mack C, Scott R. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril 1999;71:836 – 42. 13. Rienzi L, Nagy ZP, Ubaldi F, Iacobelli M, Anniballo R, Tesarik J, et al. Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged. Fertil Steril 2002;77:1196 – 201. 14. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP. Immunosuppresion supports implantation of zona pellucida dissected human embryos. Fertil Steril 1990;53:662–5.
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