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Laser assisted hatching in good prognosis patients undergoing in vitro fertilization-embryo transfer: a randomized controlled trial
Laser assisted hatching in good prognosis patients undergoing in vitro fertilization-embryo transfer: a randomized controlled trial Arthur W. Sagoskin, M.D.,a Michael J. Levy, M.D.,a Michael J. Tucker, Ph.D.,a Kevin S. Richter, Ph.D.,a and Eric A. Widra, M.D.a,b a
Shady Grove Fertility Reproductive Science Center, Rockville, Maryland, and Obstetrics and Gynecology, Washington, D.C.
b
Georgetown University Department of
Objective: To evaluate whether assisted hatching improves clinical outcomes of embryo transfers to good prognosis patients, defined as patients ⱕ39 years with normal follicle-stimulating hormone (FSH) and E2 levels, no more than one previous unsuccessful cycle of in vitro fertilization (IVF)– embryo transfer, and good embryo quality. Design: Prospective randomized controlled trial. Setting: Private assisted reproductive technology (ART) center. Patient(s): One hundred ninety-nine good prognosis patients undergoing IVF– embryo transfer. Intervention(s): In vitro fertilization followed by embryo transfer on day 3 after oocyte retrieval with or without assisted hatching using a 1,480-nm wavelength infrared laser. Main Outcome Measure(s): Clinical intrauterine pregnancy, spontaneous pregnancy loss, and live birth. Result(s): Rates of clinical intrauterine pregnancy with fetal cardiac activity (53% vs. 54% per cycle), spontaneous pregnancy loss (13% vs. 16% per pregnancy), and live birth (47% vs. 46% per cycle) were very similar between treatment cycles with laser-assisted hatching and control cycles in which embryos were transferred without assisted hatching. There were no significant differences between treatment and control groups in any measured clinical outcome parameters. Conclusion(s): Assisted hatching does not improve clinical outcomes among good prognosis patients. (Fertil Steril威 2007;87:283–7. ©2007 by American Society for Reproductive Medicine.) Key Words: In vitro fertilization, embryo transfer, assisted hatching, laser micromanipulation, good prognosis, implantation, pregnancy, live birth
The ability of a blastocyst to hatch, or escape, from the zona pellucida (ZP) that surrounds and protects the embryo during its first few days of development is one of many critical events that must occur for successful reproduction. Implantation of the embryo in the uterine lining is impossible unless hatching occurs. It has been suggested that impaired hatching of embryos resulting from IVF contributes to low implantation rates (1). Intrinsic factors such as age (2), basal FSH and diagnosis (3), the altered hormonal environment to which maturing oocytes are exposed during ovarian stimulation (3, 4), or the artificial conditions of the in vitro culture environment (5–7), may induce abnormal thickening or hardening of the ZP, making hatching more difficult. In vitro culture conditions may also adversely effect quantitative or qualitative trophectoderm-produced zona lysin secretion that has been proposed as the primary mechanism of blastocyst hatching (8). Intrinsic variation in lysin secretion independent of Received March 8, 2006; revised and accepted July 5, 2006. Presented in part at the 59th Annual Meeting of the Society for Reproductive Medicine, San Antonio, TX, October 11–15, 2003. Reprint requests: Kevin S. Richter, Ph.D., Shady Grove Fertility Reproductive Science Center, 15001 Shady Grove Road, Suite 400, Rockville, MD 20850 (FAX: 301-340-0623; E-mail: kevin.richter@integramed. com.
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assisted reproduction protocols may also contribute to the inability of some blastocysts to successfully hatch. Assisted hatching (AH), in which the ZP is artificially breached or thinned to facilitate hatching, has been proposed as a way of overcoming these intrinsic or assisted reproductive technology (ART)-induced barriers to hatching of otherwise viable embryos (1). Studies using animal models have demonstrated that AH can significantly increase rates of embryo hatching (9 –12). Assisted hatching has also been shown to significantly increase hatching rates of human embryos (13–16). Studies using animal models have also found that AH results in earlier hatching than occurs in unmanipulated embryos (8, 9). Data from patients undergoing IVF demonstrated that hCG is detectable in maternal serum earlier when AH is performed than when it is not (17), suggesting that human embryos also hatch earlier when assisted. Facilitation of earlier embryonic hatching may be particularly important given that the short window of endometrial receptivity appears to be shifted 1–2 days earlier in cycles with ovarian stimulation for ART compared to natural cycles (18 –20). A variety of different micromanipulation techniques, including mechanical partial zona dissection (1, 21, 22), zona drilling using acidic Tyrode’s solution (23, 24), laser drilling
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(12, 25–27), and piezo-micromanipulation (28) have been reported to improve ART outcomes among select groups of patients. Two recent meta-analyses of studies evaluating potential benefits of AH reported significant heterogeneity among study results (29, 30), suggesting that effects of AH may differ depending on patient characteristics. Both concluded that there is strong evidence that AH increases pregnancy rates (PR) among patients with a history of previous IVF failures. However, it remains uncertain whether AH is beneficial to other patients. Other patient populations that have been reported to benefit from AH include patients whose embryos have thick zonae and patients with elevated FSH (23), older patients (21, 24, 31–33), and patients using cryopreserved embryos (34 –37). Based on this evidence, our program routinely offers AH to patients with a history of two or more previous IVF– embryo transfer failures, patients aged 39 years or greater, and patients with a basal serum FSH concentration greater than 10 mIU/mL. However, there are indications that impaired hatching may be a direct result of ART treatment (4 – 6, 8), and thus a potentially pervasive limitation of IVF– embryo transfer implantation rates. If so, AH may also benefit patients with a better prognosis. We therefore designed this trial to test the hypothesis that AH of good quality embryos in good prognosis patients would improve PR and implantation rate. MATERIALS AND METHODS Patients undergoing IVF with cleavage stage (day 3) embryo transfer at Shady Grove Fertility Reproductive Science Center between August 2001 and March 2005 were enrolled after discussion of the study and informed written consent. Western Institutional Review Board (WIRB) approved the study protocol. This study has been registered with ClinicalTrials.gov [NCT00120549]. Inclusion criteria included first or second autologous IVF– embryo transfer cycles, maximum female age 39 years, maximum baseline FSH 10 mIU/mL, maximum baseline E2 75 pg/mL, ovulatory menstrual cycles, no uterine abnormality or communicating hydrosalpinx, and good embryo quality. Good embryo quality was defined by the presence of six to eight cells, and a maximum of 20% fragmentation on day 3. Eligible diagnoses included tubal disease (excluding communicating hydrosalpinx), endometriosis, male factor infertlity, or unexplained infertility. Patients diagnosed with diminished ovarian reserve, polycystic ovarian syndrome (PCOS), uterine or egg factor infertility were not eligible. Patients with more than one previous unsuccessful IVF cycle were also excluded. Enrolled patients were randomly assigned to either the treatment group (laser-assisted hatching [laser-AH]) or the control group (no laser-AH). Treatment assignments were determined by a computer-generated randomized series in a 2:1 ratio of treatments to controls. 284
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Assisted embryo hatching was performed using a 1,480-nm wavelength infrared laser (Zilos Laser System, Hamilton Thorne Research, Beverly, MA). An approximately 20 m defect was created in the ZP before transfer in the treatment group, whereas those in the control group were transferred with no laser-AH. The laser was set to deliver a 500-sec pulse of energy at 100% power, such that by targeting a suitable area of the ZP with an area of perivitelline space beneath, a suitable 20-m hole could be drilled with 6 –10 bursts of the laser. Embryo transfer was undertaken with either of two soft compound catheters (Tucker Embryo Catheter, Fertility Technology Resource Inc, Marietta, GA; Wallace Embryo Replacement Catheter, Irvine Scientific, Santa Ana, CA) under ultrasound guidance. Implantation and pregnancy outcomes were determined based on ultrasound identification of fetal cardiac activity 4 – 6 weeks after embryo transfer. Cycle characteristics and clinical outcomes were compared between treatment and control groups by t-test (numerical variables) or 2 (categorical variables). The sample size was calculated based on a  ⫽ 0.08 and an ␣ ⫽ 0.05 with a predicted difference in implantation rate of 8% between treatments and controls. RESULTS A total of 203 patients were consented and enrolled in the study. Three patients were excluded from subsequent analysis due to violations of inclusion criteria: elevated FSH (n ⫽ 1); advanced maternal age (n ⫽ 1); or poor embryo quality (n ⫽ 1). One patient was lost to follow-up and had no pregnancy outcome data available and was therefore excluded from analysis. Patient and cycle characteristics are compared among the 199 remaining patients in Table 1. Mean patient age was 34 years. Mean FSH was 6.6 mIU/mL, and mean E2 was 34.7 pg/mL. On day 3 after oocyte retrieval, before embryo transfer, mean cell number among transferred embryos was 7.6, with 2.9% fragmentation. A mean of 2.1 embryos were transferred per cycle. Treatment and control groups were comparable in terms of age, baseline FSH and E2, proportion of cycles with intracytoplasmic sperm injection (ICSI), cell number and fragmentation on day 3, and the number of embryos transferred. Pregnancies with fetal cardiac activity occurred in 53.8% of all treatment cycles, with an overall implantation rate of 34.0% of transferred embryos. Fifteen pregnancies (14.0%) ended in spontaneous pregnancy loss, and 92 ended in live birth (46.2% per cycle). Rates of pregnancy, implantation, spontaneous pregnancy loss, and live birth were all very similar between treatment and control cycles. There were no statistically significant differences between the treatment and control groups in any of the examined variables.
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TABLE 1 Cycle characteristics and clinical outcomes compared between cycles with assisted hatching (treatment) and those without assisted hatching (control).
Number enrolled Number analyzed Patient agea Baseline FSHa Baseline E2a ICSI Day 3 cell numbera Day 3 fragmentation (%)a Number of embryos Embryos per cyclea Positive hCG Gestational sac Fetal cardiac activity Implantation (fca/embryo) Multiple fca Spontaneous abortion Live birth per cycle
Treatment (hatched)
Control (unhatched)
121 118 34.0 ⫾ 3.3 6.6 ⫾ 1.5 35.3 ⫾ 15.6 57/118 (48.3%) 7.6 ⫾ 0.6 3.3 ⫾ 3.6 254 2.2 ⫾ 0.4 73/118 (61.9%) 64/118 (54.2%) 63/118 (53.4%) 84/254 (33.1%) 21/63 (33.3%) 8/63 (12.7%) 55/118 (46.6%)
82 81 34.0 ⫾ 3.2 6.6 ⫾ 1.6 33.7 ⫾ 14.1 38/81 (46.9%) 7.7 ⫾ 0.8 2.5 ⫾ 3.0 170 2.1 ⫾ 0.3 52/81 (64.2%) 45/81 (55.6%) 44/81 (54.3%) 60/170 (35.3%) 16/44 (36.4%) 7/44 (15.9) 37/81 (45.7%)
P value
.89 .92 .51 .85 .44 .15 .30 .75 .87 .92 .74 .91 .64 .90
Note: fca ⫽ fetal cardiac activity. a Values are means plus or minus one standard deviation. Sagoskin. Assisted hatching in good prognosis patients. Fertil Steril 2007.
DISCUSSION Many studies have evaluated the effects of AH on implantation rates of embryos transferred after IVF. However, more than 15 years after the introduction of AH into the practice of assisted reproduction, the indications for its use still remain poorly defined.
have reported significantly improved implantation rate and PR (21, 24, 31, 33), but other investigators have found no benefit (40 – 43). One study reported no benefit for AH among patients older than 36 years, but increased implantation and pregnancy with AH among patient 36 years or younger (27).
Some studies have reported significant improvements in implantation rate and PR with the application of AH to treatment cycles by “poor prognosis” patients, defined by a history of repeated IVF– embryo transfer failures, elevated FSH, or advanced age (32, 38). However, pooled analysis of such heterogeneous populations leaves ambiguity regarding whether all of these different reasons for a poor prognosis are responsive to AH. A more recent randomized study reported no benefit for AH among patients classified as poor prognosis because of either advanced age or elevated FSH (39).
An early randomized trial found that AH significantly improved implantation rate and PR among patients with a day 3 basal FSH ⬎15 mIU/mL (23). No study since has investigated AH among patients designated as poor prognosis specifically because of elevated FSH level alone. Several studies have investigated AH among cycles in which cryopreserved embryos were transferred. Some of these studies have suggested that AH increases implantation and pregnancy among this patient population (34 –37). However, other studies have reported that there is no benefit from AH of cryopreserved embryos (39, 44). Embryos with thick ZP reportedly benefit from AH (23), although this claim has been refuted (27, 42). There is also evidence that AH improves outcomes of transfers of later-developing blastocysts (45), and of embryos with zonas that were difficult to penetrate during ICSI (46).
The population for whom the benefits of AH are best documented are patients with a history of repeated IVF– embryo transfer without successful implantation. Many studies have reported significantly improved implantation rate and PR for these patients (21, 22, 24 –26, 28). Two recent meta-analyses found that there was conclusive evidence for improved success with AH among patients with repeat IVF failures (29, 30), but uncertainty regarding other patient populations. One of these meta-analyses reported a nearly significant trend toward improved outcomes among older women (29). Several studies of AH among older patients Fertility and Sterility姞
Few studies have investigated AH among patients with a relatively good prognosis. Studies of unselected populations suggest that the benefits of AH may not be universal. Antinori et al. (25) reported significant improvement in implantation and pregnancy among patients undergoing their first 285
IVF cycle. However, other randomized studies of unselected patients found no benefit from AH (47, 48). Cohen et al. (23) failed to demonstrate a significant benefit of nonselective AH in a randomized study of patients with normal day 3 basal FSH levels. Only one previous study specifically addressed good prognosis patients, defined as either new patients 30 years or younger with normal FSH, endometrial cavity, and semen parameters, or patients 35 years or younger with prior IVF experience with good fertilization and at least six embryos (49). No benefit to AH was found, although statistical power was very limited, with AH performed in fewer than 20 cycles. The present study is the first prospective randomized trial of its size to evaluate AH among selected patients with a good prognosis, defined as patients 39 years or younger with normal FSH and E2, no more than one previous unsuccessful cycle of IVF– embryo transfer, and good embryo quality on day 3 (the day when transfers were performed). Results indicate no benefit for AH among this patient population. Rates of implantation, pregnancy, spontaneous pregnancy loss, and live birth were all very similar regardless of whether or not embryos underwent AH before transfer. Although AH did not improve treatment outcomes, there was also no evidence that the laser-AH procedure harmed embryos or reduced their developmental potential, with implantation, pregnancy, and delivery rates as high in the AH group as in the control group. Studies of mouse embryos, reporting equivalent preimplantation development, equivalent rates of implantation and birth, and anatomically and immunohistochemically normal offspring with normal reproductive capacity after laser drilling of the ZP compared to unmanipulated controls, have also indicated that laser drilling of the ZP is not harmful (50, 51). A follow-up of 143 children born after laser-AH found no increase in major or minor congenital malformations or chromosomal aberrations compared to the general population (52). Few studies have compared different methods of AH, but at least two studies have suggested that laser drilling is a safer alternative to chemical drilling with acid Tyrode’s solution (53, 54). We chose to use the laser for AH in the manner described, as it allowed the most rapid and apparently least invasive approach to breaching the ZP. It is a technique that is not only easy to master, but also can be consistently applied in an easily quantifiable way. This study thus confirms the safety of the laser-AH procedure. However, it suggests that there is no benefit to performing AH on embryos of patients with a good prognosis. It is therefore recommended that AH be reserved for poor prognosis conditions for which there is more conclusive evidence of improved treatment outcomes. REFERENCES 1. 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.
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2. Garside WT, Loret de Mola JR, Bucci JA, Tureck RW, Heyner S. Sequential analysis of zona thickness during in vitro culture of human zygotes: correlation with embryo quality, age, and implantation. Mol Reprod Dev 1997;47:99 –104. 3. Loret De Mola JR, Garside WT, Bucci J, Tureck RW, Heyner S. Analysis of the human zona pellucida during culture: correlation with diagnosis and the preovulatory hormonal environment. J Assist Reprod Genet 1997;14:332– 6. 4. Bertrand E, Van den Bergh M, Englert Y. Clinical parameters influencing human zona pellucida thickness. Fertil Steril 1996;66:408 –11. 5. De Felici M, Siracusa G. “Spontaneous” hardening of the zona pellucida of mouse oocytes during in vitro cultrue. Gamete Res 1982;6: 107–13. 6. De Felici M, Salustri A, Siracusa G. “Spontaneous” hardening of the zona pellucida of mouse oocyte during in vitro culture. II. The effect of follicular fluid and glycosaminoglycans. Gamete Res 1982;12:227–35. 7. Zhang X, Rutledge J, Armstrong DT. Studies on zona hardening in rat oocytes that are matured in vitro in a serum-free medium. Mol Reprod Dev 1991;28:292– 6. 8. Schiewe MC, Hazeleger NL, Sclimenti C, Balmaceda JP. Physiological characterization of blastocyst hatching mechanisms by use of a mouse antihatching model. Fertil Steril 1995;63:288 –94. 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. Khalifa EA, Tucker MJ, Hunt P. Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse. Hum Reprod 1992;7:532– 6. 11. Alikani M, Cohen J. Micromanipulation of cleaved embryos cultured in protein-free medium: a mouse model for assisted hatching. J Exp Zool 1992;263:458 – 63. 12. 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. 13. Dokras A, Ross C, Gosden B, Sargent IL, Barlow DH. Micromanipulation of human embryos to assist hatching. Fertil Steril 1994;61: 514 –20. 14. Mandelbaum J, Plachot M, Junca AM, Salat-Baroux J, Belaisch-Allart J, Cohen J. The effects of partial zona dissection on in-vitro development and hatching of human cryopreserved embryos. Hum Reprod 1994;9(Suppl 4):39. 15. Nakayama T, Fujiwara H, Tastumi K, Fujita K, Higuchi T, Mori T. A new assisted hatching technique using a piezo-micromanipulator. Fertil Steril 1998;69:784 – 8. 16. Blake DA, Forsberg AS, Johansson BR, Wikland M. Laser zona pellucida thinning—an alternative approach to assisted hatching. Hum Reprod 2001;16:1959 – 64. 17. Liu HC, Cohen J, Alikani M, Noyes N, Rosenwaks Z. Assisted hatching facilitates earlier implantation. Fertil Steril 1993;60:871–5. 18. Develioglu OH, Hsiu JG, Nikas G, Toner JP, Oehninger S, Jones HW, Jr. Endometrial estrogen and progesterone receptor and pinopode expression in stimulated cycles of oocyte donors. Fertil Steril 1999;71: 1040 –7. 19. Nikas G, Develioglu OH, Toner JP, Jones HW, Jr. Endometrial pinopodes indicate a shift in the window of receptivity in IVF cycles. Hum Reprod 1999;14:787–92. 20. Mirkin S, Nikas G, Hsiu JG, Diaz J, Oehninger S. Gene expression profiles and structural/functional features of the peri-implantation endometrium in natural and gonadotropin-stimulated cycles. J Clin Endocrinol Metab 2004;89:5742–52. 21. 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;63:838 – 41. 22. Chao KH, Chen SU, Chen HF, Wu MY, Yang YS, Ho HN. Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF) – embryo transfer (ET), but not that of IVF–tubal ET in patients with repeated IVF failures. Fertil Steril 1997;67:904 – 8.
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23. 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;7:685–91. 24. Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Aicardi G, Montanaro N. Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching. Hum Reprod 1998;13:1331–5. 25. Antinori S, Panci C, Selman HA, Caffa B, Dani G, Versaci C. Zona thinning with the use of laser: a new approach to assisted hatching in humans. Hum Reprod 1996;11:590 – 4. 26. Antinori S, Selman HA, Caffa B, Panci C, Dani GL, Versaci C. Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy. Hum Reprod 1996;11:2488 –92. 27. Ali J, Rahbar S, Burjaq H, Sultan AM, Al Flamerzi M, Shahata MA. Routine laser assisted hatching results in significantly increased clincal pregnancies. J Assist Reprod Genet 2003;20:177– 81. 28. Nakayama T, Fujiwara H, Yamada S, Tastumi K, Honda T, Fujii S. Clinical application of a new assisted hatching method using a piezomicromanipulator for morphologically low-quality embryos in poorprognosis infertile patients. Fertil Steril 1999;71:1014 – 8. 29. Edi-Osagie E, Hooper L, Seif MW. The impact of assisted hatching on live birth rates and outcomes of assisted conception: a systematic review. Hum Reprod 2003;18:1828 –35. 30. Sallam HN, Sadek SS, Agameya AF. Assisted hatching—a metaanalysis of randomized controlled trials. J Assist Reprod Genet 2003; 20:332– 42. 31. Schoolcraft WB, Schlenker T, Jones GS, Jones HW, Jr. In vitro fertilization in women age 40 and older: the impact of assisted hatching. J Assist Reprod Genet 1995;12:581– 4. 32. Meldrum DR, Wisot A, Yee B, Garzo G, Yeo L, Hamilton F. Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozygotic twinning. J Assist Reprod Genet 1998;15:418 –21. 33. Montag M, van der Ven H. Laser-assisted hatching in assisted reproduction. Croat Med J 1999;40:398 – 403. 34. Tucker MJ, Cohen J, Massey JB, Mayer MP, Wiker SR, Wright G. Partial dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am J Obstet Gynecol 1991;165:341–5. 35. 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;65:254 –7. 36. Tao J, Tamis R. Application of assisted hatching for 2-day-old, frozenthawed embryo transfer in a poor-prognosis population. J Assist Reprod Genet 1997;14:128 –30. 37. Gabrielsen A, Agerholm I, Toft B, Hald F, Petersen K, Aagaard J, et al. Assisted hatching improves implantation rates on cryopreservedthawed embryos. A randomized prospective study. Hum Reprod 2004; 19:2258 – 62. 38. Schoolcraft WB, Schlenker T, Gee M, Jones GS, Jones HW, Jr. Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril 1994;62:551– 4. 39. Primi MP, Senn A, Montag M, Van der Ven H, Mandelbaum J, Veiga A, et al. A European multicentre prospective randomized study to assess the use of assisted hatching with a diode laser and the benefit of an immunosuppressive/antibiotic treatment in different patient populations. Hum Reprod 2004;19:2325–33.
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40. 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;12:317–20. 41. 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 1998;13:409 –13. 42. Edirisinghe WR, Ahnonkitpanit V, Promviengchai S, Suwajanakorn S, Pruksananonda K, Chinpilas V, et al. A study failing to determine significant benefits from assisted hatching: patients selected for advanced age, zonal thickness of embryos, and previous failed attempts. J Assist Reprod Genet 1999;16:294 –301. 43. Horng SG, Chang CL, Wu HM, Wang CW, Cheng CK, Huang HY, et al. Laser-assisted hatching of embryos in women of advanced age after in vitro fertilization: a preliminary report. Chang Gung Med J 2002; 25:531–7. 44. Ng EH, Naveed F, Lau EY, Yeung WS, Chan CC, Tang OS, et al. A randomized double-blind controlled study of the efficacy of laserassisted hatching on implantation and pregnancy rates of frozen-thawed embryo transfer at the cleavage stage. Hum Reprod 2005;20:979 – 85. 45. Tucker M, Graham J, Han T, Levy M, Sagoskin A, Stillman R. Enhanced implantation of day-6 blastocysts following assisted hatching with acidic tyrode’s medium. Fertil Steril 1999;72:s20 –1. 46. Ebner T, Moser M, Yaman C, Sommergruber M, Hartl J, Jesacher K, et al. Prospective hatching of embryos developed from oocytes exhibiting difficult oolemma penetration during ICSI. Hum Reprod 2002;17:1317–20. 47. 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;13:19 –22. 48. Tucker MJ, Morton PC, Wright G, Ingargiola PE, 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;11:2434 –7. 49. Hurst BS, Tucker KE, Awoniyi CA, Schlaff WD. Assisted hatching does not enhance IVF success in good-prognosis patients. J Assist Reprod Genet 1998;15:62– 4. 50. Germond M, Nocera D, Senn A, Rink K, Delacretaz G, Fakan S. Microdissection of mouse and human zona pellucida using a 1.48microns diode laser beam: efficacy and safety of the procedure. Fertil Steril 1995;64:604 –11. 51. Germond M, Nocera D, Senn A, Rink K, Delacretaz G, Pedrazzini T, et al. Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 microm diode laser beam. Hum Reprod 1996;11:1043– 8. 52. Kanyo K, Konc J. A follow-up study of children born after diode laser assisted hatching. Eur J Obstet Gynecol Reprod Biol 2003;110:176 – 80. 53. Chatzimeletiou K, Picton HM, Handyside AH. Use of a non-contact, infrared laser for zona drilling of mouse embryos: assessment of immediate effects on blastomere viability. Reprod Biomed Online 2001; 2:178 – 87. 54. Hsieh YY, Huang CC, Cheng TC, Chang CC, Tsai HD, Lee MS. Laser-assisted hatching of embryos is better than the chemical method for enhancing the pregnancy rate in women with advanced age. Fertil Steril 2002;78:179 – 82.
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Shady Grove Fertility Reproductive Science Center, Rockville, Maryland, and Obstetrics and Gynecology, Washington, D.C.
b
Georgetown University Department of
Objective: To evaluate whether assisted hatching improves clinical outcomes of embryo transfers to good prognosis patients, defined as patients ⱕ39 years with normal follicle-stimulating hormone (FSH) and E2 levels, no more than one previous unsuccessful cycle of in vitro fertilization (IVF)– embryo transfer, and good embryo quality. Design: Prospective randomized controlled trial. Setting: Private assisted reproductive technology (ART) center. Patient(s): One hundred ninety-nine good prognosis patients undergoing IVF– embryo transfer. Intervention(s): In vitro fertilization followed by embryo transfer on day 3 after oocyte retrieval with or without assisted hatching using a 1,480-nm wavelength infrared laser. Main Outcome Measure(s): Clinical intrauterine pregnancy, spontaneous pregnancy loss, and live birth. Result(s): Rates of clinical intrauterine pregnancy with fetal cardiac activity (53% vs. 54% per cycle), spontaneous pregnancy loss (13% vs. 16% per pregnancy), and live birth (47% vs. 46% per cycle) were very similar between treatment cycles with laser-assisted hatching and control cycles in which embryos were transferred without assisted hatching. There were no significant differences between treatment and control groups in any measured clinical outcome parameters. Conclusion(s): Assisted hatching does not improve clinical outcomes among good prognosis patients. (Fertil Steril威 2007;87:283–7. ©2007 by American Society for Reproductive Medicine.) Key Words: In vitro fertilization, embryo transfer, assisted hatching, laser micromanipulation, good prognosis, implantation, pregnancy, live birth
The ability of a blastocyst to hatch, or escape, from the zona pellucida (ZP) that surrounds and protects the embryo during its first few days of development is one of many critical events that must occur for successful reproduction. Implantation of the embryo in the uterine lining is impossible unless hatching occurs. It has been suggested that impaired hatching of embryos resulting from IVF contributes to low implantation rates (1). Intrinsic factors such as age (2), basal FSH and diagnosis (3), the altered hormonal environment to which maturing oocytes are exposed during ovarian stimulation (3, 4), or the artificial conditions of the in vitro culture environment (5–7), may induce abnormal thickening or hardening of the ZP, making hatching more difficult. In vitro culture conditions may also adversely effect quantitative or qualitative trophectoderm-produced zona lysin secretion that has been proposed as the primary mechanism of blastocyst hatching (8). Intrinsic variation in lysin secretion independent of Received March 8, 2006; revised and accepted July 5, 2006. Presented in part at the 59th Annual Meeting of the Society for Reproductive Medicine, San Antonio, TX, October 11–15, 2003. Reprint requests: Kevin S. Richter, Ph.D., Shady Grove Fertility Reproductive Science Center, 15001 Shady Grove Road, Suite 400, Rockville, MD 20850 (FAX: 301-340-0623; E-mail: kevin.richter@integramed. com.
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assisted reproduction protocols may also contribute to the inability of some blastocysts to successfully hatch. Assisted hatching (AH), in which the ZP is artificially breached or thinned to facilitate hatching, has been proposed as a way of overcoming these intrinsic or assisted reproductive technology (ART)-induced barriers to hatching of otherwise viable embryos (1). Studies using animal models have demonstrated that AH can significantly increase rates of embryo hatching (9 –12). Assisted hatching has also been shown to significantly increase hatching rates of human embryos (13–16). Studies using animal models have also found that AH results in earlier hatching than occurs in unmanipulated embryos (8, 9). Data from patients undergoing IVF demonstrated that hCG is detectable in maternal serum earlier when AH is performed than when it is not (17), suggesting that human embryos also hatch earlier when assisted. Facilitation of earlier embryonic hatching may be particularly important given that the short window of endometrial receptivity appears to be shifted 1–2 days earlier in cycles with ovarian stimulation for ART compared to natural cycles (18 –20). A variety of different micromanipulation techniques, including mechanical partial zona dissection (1, 21, 22), zona drilling using acidic Tyrode’s solution (23, 24), laser drilling
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(12, 25–27), and piezo-micromanipulation (28) have been reported to improve ART outcomes among select groups of patients. Two recent meta-analyses of studies evaluating potential benefits of AH reported significant heterogeneity among study results (29, 30), suggesting that effects of AH may differ depending on patient characteristics. Both concluded that there is strong evidence that AH increases pregnancy rates (PR) among patients with a history of previous IVF failures. However, it remains uncertain whether AH is beneficial to other patients. Other patient populations that have been reported to benefit from AH include patients whose embryos have thick zonae and patients with elevated FSH (23), older patients (21, 24, 31–33), and patients using cryopreserved embryos (34 –37). Based on this evidence, our program routinely offers AH to patients with a history of two or more previous IVF– embryo transfer failures, patients aged 39 years or greater, and patients with a basal serum FSH concentration greater than 10 mIU/mL. However, there are indications that impaired hatching may be a direct result of ART treatment (4 – 6, 8), and thus a potentially pervasive limitation of IVF– embryo transfer implantation rates. If so, AH may also benefit patients with a better prognosis. We therefore designed this trial to test the hypothesis that AH of good quality embryos in good prognosis patients would improve PR and implantation rate. MATERIALS AND METHODS Patients undergoing IVF with cleavage stage (day 3) embryo transfer at Shady Grove Fertility Reproductive Science Center between August 2001 and March 2005 were enrolled after discussion of the study and informed written consent. Western Institutional Review Board (WIRB) approved the study protocol. This study has been registered with ClinicalTrials.gov [NCT00120549]. Inclusion criteria included first or second autologous IVF– embryo transfer cycles, maximum female age 39 years, maximum baseline FSH 10 mIU/mL, maximum baseline E2 75 pg/mL, ovulatory menstrual cycles, no uterine abnormality or communicating hydrosalpinx, and good embryo quality. Good embryo quality was defined by the presence of six to eight cells, and a maximum of 20% fragmentation on day 3. Eligible diagnoses included tubal disease (excluding communicating hydrosalpinx), endometriosis, male factor infertlity, or unexplained infertility. Patients diagnosed with diminished ovarian reserve, polycystic ovarian syndrome (PCOS), uterine or egg factor infertility were not eligible. Patients with more than one previous unsuccessful IVF cycle were also excluded. Enrolled patients were randomly assigned to either the treatment group (laser-assisted hatching [laser-AH]) or the control group (no laser-AH). Treatment assignments were determined by a computer-generated randomized series in a 2:1 ratio of treatments to controls. 284
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Assisted embryo hatching was performed using a 1,480-nm wavelength infrared laser (Zilos Laser System, Hamilton Thorne Research, Beverly, MA). An approximately 20 m defect was created in the ZP before transfer in the treatment group, whereas those in the control group were transferred with no laser-AH. The laser was set to deliver a 500-sec pulse of energy at 100% power, such that by targeting a suitable area of the ZP with an area of perivitelline space beneath, a suitable 20-m hole could be drilled with 6 –10 bursts of the laser. Embryo transfer was undertaken with either of two soft compound catheters (Tucker Embryo Catheter, Fertility Technology Resource Inc, Marietta, GA; Wallace Embryo Replacement Catheter, Irvine Scientific, Santa Ana, CA) under ultrasound guidance. Implantation and pregnancy outcomes were determined based on ultrasound identification of fetal cardiac activity 4 – 6 weeks after embryo transfer. Cycle characteristics and clinical outcomes were compared between treatment and control groups by t-test (numerical variables) or 2 (categorical variables). The sample size was calculated based on a  ⫽ 0.08 and an ␣ ⫽ 0.05 with a predicted difference in implantation rate of 8% between treatments and controls. RESULTS A total of 203 patients were consented and enrolled in the study. Three patients were excluded from subsequent analysis due to violations of inclusion criteria: elevated FSH (n ⫽ 1); advanced maternal age (n ⫽ 1); or poor embryo quality (n ⫽ 1). One patient was lost to follow-up and had no pregnancy outcome data available and was therefore excluded from analysis. Patient and cycle characteristics are compared among the 199 remaining patients in Table 1. Mean patient age was 34 years. Mean FSH was 6.6 mIU/mL, and mean E2 was 34.7 pg/mL. On day 3 after oocyte retrieval, before embryo transfer, mean cell number among transferred embryos was 7.6, with 2.9% fragmentation. A mean of 2.1 embryos were transferred per cycle. Treatment and control groups were comparable in terms of age, baseline FSH and E2, proportion of cycles with intracytoplasmic sperm injection (ICSI), cell number and fragmentation on day 3, and the number of embryos transferred. Pregnancies with fetal cardiac activity occurred in 53.8% of all treatment cycles, with an overall implantation rate of 34.0% of transferred embryos. Fifteen pregnancies (14.0%) ended in spontaneous pregnancy loss, and 92 ended in live birth (46.2% per cycle). Rates of pregnancy, implantation, spontaneous pregnancy loss, and live birth were all very similar between treatment and control cycles. There were no statistically significant differences between the treatment and control groups in any of the examined variables.
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TABLE 1 Cycle characteristics and clinical outcomes compared between cycles with assisted hatching (treatment) and those without assisted hatching (control).
Number enrolled Number analyzed Patient agea Baseline FSHa Baseline E2a ICSI Day 3 cell numbera Day 3 fragmentation (%)a Number of embryos Embryos per cyclea Positive hCG Gestational sac Fetal cardiac activity Implantation (fca/embryo) Multiple fca Spontaneous abortion Live birth per cycle
Treatment (hatched)
Control (unhatched)
121 118 34.0 ⫾ 3.3 6.6 ⫾ 1.5 35.3 ⫾ 15.6 57/118 (48.3%) 7.6 ⫾ 0.6 3.3 ⫾ 3.6 254 2.2 ⫾ 0.4 73/118 (61.9%) 64/118 (54.2%) 63/118 (53.4%) 84/254 (33.1%) 21/63 (33.3%) 8/63 (12.7%) 55/118 (46.6%)
82 81 34.0 ⫾ 3.2 6.6 ⫾ 1.6 33.7 ⫾ 14.1 38/81 (46.9%) 7.7 ⫾ 0.8 2.5 ⫾ 3.0 170 2.1 ⫾ 0.3 52/81 (64.2%) 45/81 (55.6%) 44/81 (54.3%) 60/170 (35.3%) 16/44 (36.4%) 7/44 (15.9) 37/81 (45.7%)
P value
.89 .92 .51 .85 .44 .15 .30 .75 .87 .92 .74 .91 .64 .90
Note: fca ⫽ fetal cardiac activity. a Values are means plus or minus one standard deviation. Sagoskin. Assisted hatching in good prognosis patients. Fertil Steril 2007.
DISCUSSION Many studies have evaluated the effects of AH on implantation rates of embryos transferred after IVF. However, more than 15 years after the introduction of AH into the practice of assisted reproduction, the indications for its use still remain poorly defined.
have reported significantly improved implantation rate and PR (21, 24, 31, 33), but other investigators have found no benefit (40 – 43). One study reported no benefit for AH among patients older than 36 years, but increased implantation and pregnancy with AH among patient 36 years or younger (27).
Some studies have reported significant improvements in implantation rate and PR with the application of AH to treatment cycles by “poor prognosis” patients, defined by a history of repeated IVF– embryo transfer failures, elevated FSH, or advanced age (32, 38). However, pooled analysis of such heterogeneous populations leaves ambiguity regarding whether all of these different reasons for a poor prognosis are responsive to AH. A more recent randomized study reported no benefit for AH among patients classified as poor prognosis because of either advanced age or elevated FSH (39).
An early randomized trial found that AH significantly improved implantation rate and PR among patients with a day 3 basal FSH ⬎15 mIU/mL (23). No study since has investigated AH among patients designated as poor prognosis specifically because of elevated FSH level alone. Several studies have investigated AH among cycles in which cryopreserved embryos were transferred. Some of these studies have suggested that AH increases implantation and pregnancy among this patient population (34 –37). However, other studies have reported that there is no benefit from AH of cryopreserved embryos (39, 44). Embryos with thick ZP reportedly benefit from AH (23), although this claim has been refuted (27, 42). There is also evidence that AH improves outcomes of transfers of later-developing blastocysts (45), and of embryos with zonas that were difficult to penetrate during ICSI (46).
The population for whom the benefits of AH are best documented are patients with a history of repeated IVF– embryo transfer without successful implantation. Many studies have reported significantly improved implantation rate and PR for these patients (21, 22, 24 –26, 28). Two recent meta-analyses found that there was conclusive evidence for improved success with AH among patients with repeat IVF failures (29, 30), but uncertainty regarding other patient populations. One of these meta-analyses reported a nearly significant trend toward improved outcomes among older women (29). Several studies of AH among older patients Fertility and Sterility姞
Few studies have investigated AH among patients with a relatively good prognosis. Studies of unselected populations suggest that the benefits of AH may not be universal. Antinori et al. (25) reported significant improvement in implantation and pregnancy among patients undergoing their first 285
IVF cycle. However, other randomized studies of unselected patients found no benefit from AH (47, 48). Cohen et al. (23) failed to demonstrate a significant benefit of nonselective AH in a randomized study of patients with normal day 3 basal FSH levels. Only one previous study specifically addressed good prognosis patients, defined as either new patients 30 years or younger with normal FSH, endometrial cavity, and semen parameters, or patients 35 years or younger with prior IVF experience with good fertilization and at least six embryos (49). No benefit to AH was found, although statistical power was very limited, with AH performed in fewer than 20 cycles. The present study is the first prospective randomized trial of its size to evaluate AH among selected patients with a good prognosis, defined as patients 39 years or younger with normal FSH and E2, no more than one previous unsuccessful cycle of IVF– embryo transfer, and good embryo quality on day 3 (the day when transfers were performed). Results indicate no benefit for AH among this patient population. Rates of implantation, pregnancy, spontaneous pregnancy loss, and live birth were all very similar regardless of whether or not embryos underwent AH before transfer. Although AH did not improve treatment outcomes, there was also no evidence that the laser-AH procedure harmed embryos or reduced their developmental potential, with implantation, pregnancy, and delivery rates as high in the AH group as in the control group. Studies of mouse embryos, reporting equivalent preimplantation development, equivalent rates of implantation and birth, and anatomically and immunohistochemically normal offspring with normal reproductive capacity after laser drilling of the ZP compared to unmanipulated controls, have also indicated that laser drilling of the ZP is not harmful (50, 51). A follow-up of 143 children born after laser-AH found no increase in major or minor congenital malformations or chromosomal aberrations compared to the general population (52). Few studies have compared different methods of AH, but at least two studies have suggested that laser drilling is a safer alternative to chemical drilling with acid Tyrode’s solution (53, 54). We chose to use the laser for AH in the manner described, as it allowed the most rapid and apparently least invasive approach to breaching the ZP. It is a technique that is not only easy to master, but also can be consistently applied in an easily quantifiable way. This study thus confirms the safety of the laser-AH procedure. However, it suggests that there is no benefit to performing AH on embryos of patients with a good prognosis. It is therefore recommended that AH be reserved for poor prognosis conditions for which there is more conclusive evidence of improved treatment outcomes. REFERENCES 1. 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.
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40. 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;12:317–20. 41. 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 1998;13:409 –13. 42. Edirisinghe WR, Ahnonkitpanit V, Promviengchai S, Suwajanakorn S, Pruksananonda K, Chinpilas V, et al. A study failing to determine significant benefits from assisted hatching: patients selected for advanced age, zonal thickness of embryos, and previous failed attempts. J Assist Reprod Genet 1999;16:294 –301. 43. Horng SG, Chang CL, Wu HM, Wang CW, Cheng CK, Huang HY, et al. Laser-assisted hatching of embryos in women of advanced age after in vitro fertilization: a preliminary report. Chang Gung Med J 2002; 25:531–7. 44. Ng EH, Naveed F, Lau EY, Yeung WS, Chan CC, Tang OS, et al. A randomized double-blind controlled study of the efficacy of laserassisted hatching on implantation and pregnancy rates of frozen-thawed embryo transfer at the cleavage stage. Hum Reprod 2005;20:979 – 85. 45. Tucker M, Graham J, Han T, Levy M, Sagoskin A, Stillman R. Enhanced implantation of day-6 blastocysts following assisted hatching with acidic tyrode’s medium. Fertil Steril 1999;72:s20 –1. 46. Ebner T, Moser M, Yaman C, Sommergruber M, Hartl J, Jesacher K, et al. Prospective hatching of embryos developed from oocytes exhibiting difficult oolemma penetration during ICSI. Hum Reprod 2002;17:1317–20. 47. 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;13:19 –22. 48. Tucker MJ, Morton PC, Wright G, Ingargiola PE, 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;11:2434 –7. 49. Hurst BS, Tucker KE, Awoniyi CA, Schlaff WD. Assisted hatching does not enhance IVF success in good-prognosis patients. J Assist Reprod Genet 1998;15:62– 4. 50. Germond M, Nocera D, Senn A, Rink K, Delacretaz G, Fakan S. Microdissection of mouse and human zona pellucida using a 1.48microns diode laser beam: efficacy and safety of the procedure. Fertil Steril 1995;64:604 –11. 51. Germond M, Nocera D, Senn A, Rink K, Delacretaz G, Pedrazzini T, et al. Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 microm diode laser beam. Hum Reprod 1996;11:1043– 8. 52. Kanyo K, Konc J. A follow-up study of children born after diode laser assisted hatching. Eur J Obstet Gynecol Reprod Biol 2003;110:176 – 80. 53. Chatzimeletiou K, Picton HM, Handyside AH. Use of a non-contact, infrared laser for zona drilling of mouse embryos: assessment of immediate effects on blastomere viability. Reprod Biomed Online 2001; 2:178 – 87. 54. Hsieh YY, Huang CC, Cheng TC, Chang CC, Tsai HD, Lee MS. Laser-assisted hatching of embryos is better than the chemical method for enhancing the pregnancy rate in women with advanced age. Fertil Steril 2002;78:179 – 82.
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