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Clinical application of nonselective assisted hatching of human embryos*
Vol. 66, No.6, December 1996
FERTILITY AND STERILITY® Copyright
Printed on acid-free paper in U. S. A.
1996 American Society for Reproductive Medicine
Clinical application of nonselective assisted hatching of human embryos* Yunxia Hu, B.S. t David 1. Hoffman, M.D. Wayne S. Maxson, M.D. Steven J. Ory, M.D. Northwest Center for Infertility and Reproductive Endocrinology, Margate, Florida
Objective: To evaluate the effect of nonselective assisted hatching on pregnancy rate (PR) and to provide an alternative and simplified method for clinical application of assisted hatching. Design: Retrospective analysis of clinical data. Setting: Private infertility practice. Patient(s): Women from 258 consecutive stimulated IVF cycles. Intervention(s): Assisted hatching was performed on each transferred embryo regardless of patient history, embryo morphology, or other selection criteria routinely applied to many IVF programs. Main Outcome Measure(s): Pregnancy, live birth, and implantation rates. Result(s): Of 258 consecutive patients who had nonselective assisted hatching, 109 (42%) had clinical pregnancies, with 93 (36%) live births and 178 (20%) embryos implanted. Conclusion(s): Nonselective assisted hatching resulted in an acceptable PR and provided an alternative and simplified method for clinical application of assisted hatching. Fertil Steril® 1996;66:991-4 Key Words: Assisted hatching, pregnancy rate, live births, implantation rates
The potential clinical advantage of embryo hatching was first suggested after an incidental observation in the course of studies using mouse and human embryos after zona drilling with acidic Tyrode's solution, partial zona dissection, and zona chiseling method 0-3). In the course of these studies it was recognized that treated embryos began to hatch and extrusion occurred earlier. Abnormalities of the hatching process were suspected as a possible contributing factor to failed nidation. Also, some embryos became entrapped within the zona pellucida, suggesting a need for larger holes to promote more complete hatching (4).
Received March 8, 1996; revised and accepted July 11, 1996. * Presented as a poster at the European Society of Human Reproduction and Embryology, Hamburg, Germany, June 28 to July 1,1995. t Reprint requests: Yunxia Hu, B.S., Northwest Center for Infertility and Reproductive Endocrinology, 2825 North State Road 7, Suite 302, Margate, Florida 33063 (FAX: 954-972-6310). Vol. 66, No.6, December 1996
Human embryo implantation rates have been improved significantly since assisted hatching was first attempted with partial zona dissection performed on micromanipulated embryos (5). Assisted hatching performed on selected embryos enhanced implantation rates using acidic Tyrode's soluti0I1 (6). The combination of selective assisted hatching and embryo coculture also was noted to improve pregnancy rates (PRs) in a selected patient population (7). Selection criteria, primarily established by Cohen et al. (6), have been followed by many laboratories to select embryos for assisted hatching. Determining whether an embryo meets the criteria entails removing embryos from the incubator for variable time intervals to make the requisite morphological determinations and zona measurements procedures, which themselves may be detrimental to the embryos. Using assisted hatching for all patients without using selective criteria eliminates this step and might be associated with improved PRs. This report describes the experience of 258 patients consecuHu et al. Nonselective assisted hatching
991
r
tively treated with nonselective assisted hatching in 1994. A total of 895 embryos underwent assisted hatching and IVF-ET.
uterine sac at sonography and live births had gestational age of 2"028 weeks after retrieval. Nonselective Assisted Hatching
MATERIALS AND METHODS Patients and IVF Procedure
All IVF patients undergoing ET were included in this protocol regardless of prior clinical history or embryo morphology. All patients were between the ages of24 and 42 years and provided written consent to the procedure. The GnRH agonist leuprolide acetate (LA, Lupron; Tap Pharmaceuticals, Deerfield, IL) was initiated in the midluteal phase (8) or early follicular phase (flare regimen). Leuprolide acetate was continued until down-regulation was documented with serum E2 determination and ultrasound for the midluteal regimen. The flare regimen was used in patients having an intermenstrual interval> 35 days, a midluteal P level < 5 ng/mL (conversion factor to SI unit, 3.180), or an absent LH surge (9). All patients subsequently received hMG (Pergonal or Metrodin; Serono Laboratories, Norwell, MA) for ovarian stimulation. Each patient received 16 mg methylprednisolone and 1 g tetracycline daily for 4 days, commencing on the day of oocyte retrieval. Oocyte Culture
After retrieval, oocytes were cultured in human tubal fluid supplemented with 10% maternal serum (10). Donor serum or synthetic serum substitute (Irvine Scientific, Irvine, CA) was used if the female partner demonstrated antisperm antibodies. Conventional IVF insemination was used for fertilization, with the exception of subzonal insertion for 5 patients and intracytoplasmic sperm injection for 34 patients. Zygotes were cultured for 72 hours (3 days after oocyte retrieval) in 30- to 50-pL droplets of human tubal fluid supplemented with 15% maternal serum under mineral oil (Squibb, Rahway, NJ). Embryo evaluation for replacement was performed on the morning of the 3rd day after oocyte retrieval. Embryos with the highest number of blastomeres and lowest rate of fragmentation were selected for replacement when more than three embryos were available. Assisted hatching was performed on 2- to 12-cell embryos, but usually on the 4- to 8-cell stage. Embryo transfer was performed 72 to 76 hours after oocyte retrieval, usually with an Edwards-Wallace catheter (Marlow Surgical Technologies, Willoughby, OH). A biochemical pregnancy was confirmed with an increase in serum ,B-hCG noted after three consecutive determinations. Clinical pregnancy was identified by presence of an intra992
Hu et al. Nonselective assisted hatching
Assisted hatching using acidic Tyrode's solution on each embryo was performed on a prewarmed Nunc dish lid (100 X 20 mm; Nunc, Roskilde, Denmark) in a 15- to 20-pL droplet of modified phosphate-buffered saline covered with warm paraffin oil (CMS, Houston, TX). The micromanipulation procedure (6) was performed with the following modification. The embryo was held on a holding pipette (10 to 20 /.Lm inner diameter) in such a way that a large area (approximately 30 /.Lm) of the zona pellucida was thinned on the side of perivitelline space or the extracellular fragments using a preloaded Tyrode's solution (pH 2.2 to 2.5) in a hatching needle (approximately 10 /.Lm inner diameter). A small opening (approximately 5 to 7 /.Lm) was made mechanically to complete the procedure. If > 10% extracellular fragments were observed, they were removed during the assisted hatching procedure. Each embryo was manipulated individually and rinsed five times. The embryos then were cultured for up to 4 hours before replacement. Statistical Analysis X 2 tests were used to compare PRs and implantation rates between three infertility diagnosis groups and between three age categories from normal and male factor groups. A difference of P < 0.05 was considered to be significant.
RESULTS
Overall, from a total of 258 consecutive patients who were taken to retrieval, 895 assisted hatching embryos were transferred to 245 patients (13 patients did not have embryos for ET bec,ause of fertilization failure). The implantation rate per embryo (defined by the observation of cardiac activity by ultrasonography) was 20% (178/895) and an average of 3.5 ± 1.18 (mean ± SD) embryos were replaced per patient. Biochemical, clinical, live birth, and multiple live birth rates were 48% (123/258), 42% (109/258), 36% (931258), and 52% (48/93), respectively. Effects of infertility diagnosis on pregnancy and implantation are presented in Table 1. The causes of infertility were categorized into three groups. Forty-six percent of the patients (n = 258) presented in the nontubal factor group with the primary diagnosis of an elevated day 2 serum FSH level, ovulatory dysfunction, or multiple prior IVF failure. Thirty-two percent of the patients had the diagnosis of tubal factor, and 22% had a diagnosis Fertility and Sterility®
Table 1 Effect of Infertility Diagnosis on Pregnancy and Implantation Rates
No. of patients* Age (y)t Clinical Live birth Implantation
Nontubal factor
Tubal factor
Male factor
119/258 (46) 36.5 :+: 4.6 46/119 (39) 40/119 (34) 79/451 (18)
82/258 (32) 33.9 :+: 4.1 41/82 (50) 32/82 (39) 63/279 (23)
57/258 (22) 33.2 :+: 3.8 22/57 (39) 21/57 (37) 36/165 (22)
* Values in parentheses are
percentages.
t Values are means:+: SD.
of male factor (defined by sperm motility of <40% and concentration of <20 X 106 spermlmL). Of 258 retrievals, the clinical pregnancy (39%, 50%, and 39%), live birth (34%,39%, and 37%), and implantation rates (18%, 23%, and 22%) were achieved, respectively, from each group with no statistical differences observed. Further breakdown of clinical PR and live birth rate according to maternal age and the presence of nonmale or male factor are presented in Table 2. Both clinical PR and live birth rate varied according to maternal age. Pregnancy and live birth rate decreased as the maternal age increased in both the normal male and the male factor groups. In the normal male group, as the maternal age increased, the PR changed from 51 % (48/94) to 40% (26/65) and to a low of 31% (13/42); and the live birth rate changed from 41 % (39/94) to 35% (23/65) and to a low of24% (10/42), with a significant difference observed from <35 versus >38 years age category (P < 0.05). In the male factor group the same trend was observed with PRs of 45% (14/31),37% (7119), and 14% (117) (P < 0.05) and live birth rates of 45% (14/31), 37% (6119), and 14% (117)), respectively, in each age category. The male factor group did not differ significantly from the normal male group. DISCUSSION
Cohen et al. (5) suggested that assisted hatching was most effective in embryos with thick zona, patients aged >38 years, and those patients with elevated FSH levels. They also suggested that the embryos with thin zonae may be jeopardized by the assisted hatching procedure. These findings cannot be confirmed from our clinical assisted hatching application because the acceptable live birth and implantation rates had been achieved from our nonselective assisted hatching procedure. Cohen (4) suggested that hatching embryos with high implantation prognosis may cause premature hatching, which may result in trophoblast trapping and disintegration of the embryos. From our observations, hatching embryos from the tubal factor Vol. 66, No.6, December 1996
group, which have a high implantation prognosis, resulted in an acceptable implantation rate (63/279, 23%) and clinical PR (41/82, 50%). There is a concern of possible increasing the incidence of monozygotic twinning that may result from assisted hatching (1). Too narrow of an opening may cause the resulting blastocyst to separate into several pieces, which may result in monozygotic twinning. We observed two cases of monozygotic twinning after the application of nonselective assisted hatching in 258 patients (0.8%). Both of these cases resulted in live, healthy deliveries of female and male babies. Maintaining a stable environment of 5% CO 2 and 37°C are critical objectives in an IVF laboratory. When embryos are removed from the incubator for observation, they are subjected to potential changes in pH and temperature. The nonselective assisted hatching procedure eliminates the need for zona thickness measurement, patient history evaluation, and morphological evaluation before the assisted hatching procedure. Therefore, it reduces the amount of time the embryo culture dish is removed from an incubator for selection and reduces the potential for variation in these parameters. Conversely, nonselective assisted hatching subjects all transferred embryos to potential changes in these parameters so equipment and procedures must be in place to prevent this from occurring. Hatching all transferred embryos is time consuming in clinical application and may add more work for laboratory personnel, but it can be managed by experienced embryologists. Furthermore, the cost of assisted hatching is reduced by eliminating the need for equipment used to measure zona thickness. Damage to the embryos during assisted hatching may occur ifthe embryologist is inexperienced or if hatching needles are too large and do not provide adequate control of the acid solution. In this trial, no noticeable or suspected damage to blastomeres occurred during hatching of 895 embryos. This observation as well as the PRs and implantation rates resulting Table 2 Comparison of Clinical and Live Birth Rates per Retrieval by Age Between Nonmale and Male Factor Groups Clinical
Live birth
Age group
Normal male
Male factor
Normal male
Male factor
<35* 35 to 38 >38
48/94 (51)t 26/65 (40) 13/42 (31)
14/31 (45) 7/19 (37) 117 (14)
39/94 (41):1: 23/65 (35) 10/42 (24)
14/31 (45) 6/19 (37) 117 (14)
* Values in
parentheses are percentages. t Significantly different from clinical PR age > 38 years for normal male; X2 , P < 0.05. :I: Significantly different from live birth rate age> 38 years for normal male; X2, P < 0.05. Hu et al.
Nonselective assisted hatching
993
from the transfer of these embryos supports that nonselective assisted hatching can by used by experienced technicians without increasing risk to the embryos or to the overall outcome. The data presented demonstrates that nonselective assisted hatching can be used to produce acceptable PRs and live birth rates. Further investigation is still needed to determine if this procedure is a major factor in improving implantation and PRs. Acknowledgments. The authors are grateful for the support of staff at the N3 and wish to thank Sue Eager, B.S.M.T., Julie Romero Dupre, M.S., Irma Fiore, B.S.M.T., and Nancy Burozski, B.A., for technical support and Ms. Ellen Hagen, Ms. Sandra Berven, and Lisa Nichols, B.S., for preparation of the manuscript.
4. 5.
6.
7.
8.
REFERENCES 1. Malter HE, Cohen J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res 1989;24:67-80. 2. Malter HE, Cohen J. Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 1989;51:13948. 3. Cordon JW, Talansky BE. Assisted fertilization by zona drill-
994
Hu et al. Nonselective assisted hatching
9.
10.
ing: a mouse model for correction of oligospermia. J Exp Zool 1986; 239:347-54. Cohen J. Assisted hatching of human embryos. J In Vitro Fert Embryo Transf 1991;8:179-90. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer M, 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. 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. Wiemer RK, Hu Y, Cuervo M, Genetis P, Leibowitz D. The combination of coculture and selective assisted hatching: results from their clinical application. Fertil Steril 1994;61: 105-10. Droesch K, Muasher SJ, Brzyski RG, Jones GS, Simonetti S, Liu H-C, et al. Value of suppression with a gonadotropin stimulation for in vitro fertilization. Fertil Steril 1989;51: 292-7. Garcia E, Padilla SL, Bayati J, Baramki TA. Follicular phase gonadotropin-releasing hormone agonist and human gonadotropins: a better alternative for ovulation induction in in vitro fertilization. Fertil Steril 1990;53:302-5. Quinn P, Kerin F, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493-8.
Fertility and Sterility®
FERTILITY AND STERILITY® Copyright
Printed on acid-free paper in U. S. A.
1996 American Society for Reproductive Medicine
Clinical application of nonselective assisted hatching of human embryos* Yunxia Hu, B.S. t David 1. Hoffman, M.D. Wayne S. Maxson, M.D. Steven J. Ory, M.D. Northwest Center for Infertility and Reproductive Endocrinology, Margate, Florida
Objective: To evaluate the effect of nonselective assisted hatching on pregnancy rate (PR) and to provide an alternative and simplified method for clinical application of assisted hatching. Design: Retrospective analysis of clinical data. Setting: Private infertility practice. Patient(s): Women from 258 consecutive stimulated IVF cycles. Intervention(s): Assisted hatching was performed on each transferred embryo regardless of patient history, embryo morphology, or other selection criteria routinely applied to many IVF programs. Main Outcome Measure(s): Pregnancy, live birth, and implantation rates. Result(s): Of 258 consecutive patients who had nonselective assisted hatching, 109 (42%) had clinical pregnancies, with 93 (36%) live births and 178 (20%) embryos implanted. Conclusion(s): Nonselective assisted hatching resulted in an acceptable PR and provided an alternative and simplified method for clinical application of assisted hatching. Fertil Steril® 1996;66:991-4 Key Words: Assisted hatching, pregnancy rate, live births, implantation rates
The potential clinical advantage of embryo hatching was first suggested after an incidental observation in the course of studies using mouse and human embryos after zona drilling with acidic Tyrode's solution, partial zona dissection, and zona chiseling method 0-3). In the course of these studies it was recognized that treated embryos began to hatch and extrusion occurred earlier. Abnormalities of the hatching process were suspected as a possible contributing factor to failed nidation. Also, some embryos became entrapped within the zona pellucida, suggesting a need for larger holes to promote more complete hatching (4).
Received March 8, 1996; revised and accepted July 11, 1996. * Presented as a poster at the European Society of Human Reproduction and Embryology, Hamburg, Germany, June 28 to July 1,1995. t Reprint requests: Yunxia Hu, B.S., Northwest Center for Infertility and Reproductive Endocrinology, 2825 North State Road 7, Suite 302, Margate, Florida 33063 (FAX: 954-972-6310). Vol. 66, No.6, December 1996
Human embryo implantation rates have been improved significantly since assisted hatching was first attempted with partial zona dissection performed on micromanipulated embryos (5). Assisted hatching performed on selected embryos enhanced implantation rates using acidic Tyrode's soluti0I1 (6). The combination of selective assisted hatching and embryo coculture also was noted to improve pregnancy rates (PRs) in a selected patient population (7). Selection criteria, primarily established by Cohen et al. (6), have been followed by many laboratories to select embryos for assisted hatching. Determining whether an embryo meets the criteria entails removing embryos from the incubator for variable time intervals to make the requisite morphological determinations and zona measurements procedures, which themselves may be detrimental to the embryos. Using assisted hatching for all patients without using selective criteria eliminates this step and might be associated with improved PRs. This report describes the experience of 258 patients consecuHu et al. Nonselective assisted hatching
991
r
tively treated with nonselective assisted hatching in 1994. A total of 895 embryos underwent assisted hatching and IVF-ET.
uterine sac at sonography and live births had gestational age of 2"028 weeks after retrieval. Nonselective Assisted Hatching
MATERIALS AND METHODS Patients and IVF Procedure
All IVF patients undergoing ET were included in this protocol regardless of prior clinical history or embryo morphology. All patients were between the ages of24 and 42 years and provided written consent to the procedure. The GnRH agonist leuprolide acetate (LA, Lupron; Tap Pharmaceuticals, Deerfield, IL) was initiated in the midluteal phase (8) or early follicular phase (flare regimen). Leuprolide acetate was continued until down-regulation was documented with serum E2 determination and ultrasound for the midluteal regimen. The flare regimen was used in patients having an intermenstrual interval> 35 days, a midluteal P level < 5 ng/mL (conversion factor to SI unit, 3.180), or an absent LH surge (9). All patients subsequently received hMG (Pergonal or Metrodin; Serono Laboratories, Norwell, MA) for ovarian stimulation. Each patient received 16 mg methylprednisolone and 1 g tetracycline daily for 4 days, commencing on the day of oocyte retrieval. Oocyte Culture
After retrieval, oocytes were cultured in human tubal fluid supplemented with 10% maternal serum (10). Donor serum or synthetic serum substitute (Irvine Scientific, Irvine, CA) was used if the female partner demonstrated antisperm antibodies. Conventional IVF insemination was used for fertilization, with the exception of subzonal insertion for 5 patients and intracytoplasmic sperm injection for 34 patients. Zygotes were cultured for 72 hours (3 days after oocyte retrieval) in 30- to 50-pL droplets of human tubal fluid supplemented with 15% maternal serum under mineral oil (Squibb, Rahway, NJ). Embryo evaluation for replacement was performed on the morning of the 3rd day after oocyte retrieval. Embryos with the highest number of blastomeres and lowest rate of fragmentation were selected for replacement when more than three embryos were available. Assisted hatching was performed on 2- to 12-cell embryos, but usually on the 4- to 8-cell stage. Embryo transfer was performed 72 to 76 hours after oocyte retrieval, usually with an Edwards-Wallace catheter (Marlow Surgical Technologies, Willoughby, OH). A biochemical pregnancy was confirmed with an increase in serum ,B-hCG noted after three consecutive determinations. Clinical pregnancy was identified by presence of an intra992
Hu et al. Nonselective assisted hatching
Assisted hatching using acidic Tyrode's solution on each embryo was performed on a prewarmed Nunc dish lid (100 X 20 mm; Nunc, Roskilde, Denmark) in a 15- to 20-pL droplet of modified phosphate-buffered saline covered with warm paraffin oil (CMS, Houston, TX). The micromanipulation procedure (6) was performed with the following modification. The embryo was held on a holding pipette (10 to 20 /.Lm inner diameter) in such a way that a large area (approximately 30 /.Lm) of the zona pellucida was thinned on the side of perivitelline space or the extracellular fragments using a preloaded Tyrode's solution (pH 2.2 to 2.5) in a hatching needle (approximately 10 /.Lm inner diameter). A small opening (approximately 5 to 7 /.Lm) was made mechanically to complete the procedure. If > 10% extracellular fragments were observed, they were removed during the assisted hatching procedure. Each embryo was manipulated individually and rinsed five times. The embryos then were cultured for up to 4 hours before replacement. Statistical Analysis X 2 tests were used to compare PRs and implantation rates between three infertility diagnosis groups and between three age categories from normal and male factor groups. A difference of P < 0.05 was considered to be significant.
RESULTS
Overall, from a total of 258 consecutive patients who were taken to retrieval, 895 assisted hatching embryos were transferred to 245 patients (13 patients did not have embryos for ET bec,ause of fertilization failure). The implantation rate per embryo (defined by the observation of cardiac activity by ultrasonography) was 20% (178/895) and an average of 3.5 ± 1.18 (mean ± SD) embryos were replaced per patient. Biochemical, clinical, live birth, and multiple live birth rates were 48% (123/258), 42% (109/258), 36% (931258), and 52% (48/93), respectively. Effects of infertility diagnosis on pregnancy and implantation are presented in Table 1. The causes of infertility were categorized into three groups. Forty-six percent of the patients (n = 258) presented in the nontubal factor group with the primary diagnosis of an elevated day 2 serum FSH level, ovulatory dysfunction, or multiple prior IVF failure. Thirty-two percent of the patients had the diagnosis of tubal factor, and 22% had a diagnosis Fertility and Sterility®
Table 1 Effect of Infertility Diagnosis on Pregnancy and Implantation Rates
No. of patients* Age (y)t Clinical Live birth Implantation
Nontubal factor
Tubal factor
Male factor
119/258 (46) 36.5 :+: 4.6 46/119 (39) 40/119 (34) 79/451 (18)
82/258 (32) 33.9 :+: 4.1 41/82 (50) 32/82 (39) 63/279 (23)
57/258 (22) 33.2 :+: 3.8 22/57 (39) 21/57 (37) 36/165 (22)
* Values in parentheses are
percentages.
t Values are means:+: SD.
of male factor (defined by sperm motility of <40% and concentration of <20 X 106 spermlmL). Of 258 retrievals, the clinical pregnancy (39%, 50%, and 39%), live birth (34%,39%, and 37%), and implantation rates (18%, 23%, and 22%) were achieved, respectively, from each group with no statistical differences observed. Further breakdown of clinical PR and live birth rate according to maternal age and the presence of nonmale or male factor are presented in Table 2. Both clinical PR and live birth rate varied according to maternal age. Pregnancy and live birth rate decreased as the maternal age increased in both the normal male and the male factor groups. In the normal male group, as the maternal age increased, the PR changed from 51 % (48/94) to 40% (26/65) and to a low of 31% (13/42); and the live birth rate changed from 41 % (39/94) to 35% (23/65) and to a low of24% (10/42), with a significant difference observed from <35 versus >38 years age category (P < 0.05). In the male factor group the same trend was observed with PRs of 45% (14/31),37% (7119), and 14% (117) (P < 0.05) and live birth rates of 45% (14/31), 37% (6119), and 14% (117)), respectively, in each age category. The male factor group did not differ significantly from the normal male group. DISCUSSION
Cohen et al. (5) suggested that assisted hatching was most effective in embryos with thick zona, patients aged >38 years, and those patients with elevated FSH levels. They also suggested that the embryos with thin zonae may be jeopardized by the assisted hatching procedure. These findings cannot be confirmed from our clinical assisted hatching application because the acceptable live birth and implantation rates had been achieved from our nonselective assisted hatching procedure. Cohen (4) suggested that hatching embryos with high implantation prognosis may cause premature hatching, which may result in trophoblast trapping and disintegration of the embryos. From our observations, hatching embryos from the tubal factor Vol. 66, No.6, December 1996
group, which have a high implantation prognosis, resulted in an acceptable implantation rate (63/279, 23%) and clinical PR (41/82, 50%). There is a concern of possible increasing the incidence of monozygotic twinning that may result from assisted hatching (1). Too narrow of an opening may cause the resulting blastocyst to separate into several pieces, which may result in monozygotic twinning. We observed two cases of monozygotic twinning after the application of nonselective assisted hatching in 258 patients (0.8%). Both of these cases resulted in live, healthy deliveries of female and male babies. Maintaining a stable environment of 5% CO 2 and 37°C are critical objectives in an IVF laboratory. When embryos are removed from the incubator for observation, they are subjected to potential changes in pH and temperature. The nonselective assisted hatching procedure eliminates the need for zona thickness measurement, patient history evaluation, and morphological evaluation before the assisted hatching procedure. Therefore, it reduces the amount of time the embryo culture dish is removed from an incubator for selection and reduces the potential for variation in these parameters. Conversely, nonselective assisted hatching subjects all transferred embryos to potential changes in these parameters so equipment and procedures must be in place to prevent this from occurring. Hatching all transferred embryos is time consuming in clinical application and may add more work for laboratory personnel, but it can be managed by experienced embryologists. Furthermore, the cost of assisted hatching is reduced by eliminating the need for equipment used to measure zona thickness. Damage to the embryos during assisted hatching may occur ifthe embryologist is inexperienced or if hatching needles are too large and do not provide adequate control of the acid solution. In this trial, no noticeable or suspected damage to blastomeres occurred during hatching of 895 embryos. This observation as well as the PRs and implantation rates resulting Table 2 Comparison of Clinical and Live Birth Rates per Retrieval by Age Between Nonmale and Male Factor Groups Clinical
Live birth
Age group
Normal male
Male factor
Normal male
Male factor
<35* 35 to 38 >38
48/94 (51)t 26/65 (40) 13/42 (31)
14/31 (45) 7/19 (37) 117 (14)
39/94 (41):1: 23/65 (35) 10/42 (24)
14/31 (45) 6/19 (37) 117 (14)
* Values in
parentheses are percentages. t Significantly different from clinical PR age > 38 years for normal male; X2 , P < 0.05. :I: Significantly different from live birth rate age> 38 years for normal male; X2, P < 0.05. Hu et al.
Nonselective assisted hatching
993
from the transfer of these embryos supports that nonselective assisted hatching can by used by experienced technicians without increasing risk to the embryos or to the overall outcome. The data presented demonstrates that nonselective assisted hatching can be used to produce acceptable PRs and live birth rates. Further investigation is still needed to determine if this procedure is a major factor in improving implantation and PRs. Acknowledgments. The authors are grateful for the support of staff at the N3 and wish to thank Sue Eager, B.S.M.T., Julie Romero Dupre, M.S., Irma Fiore, B.S.M.T., and Nancy Burozski, B.A., for technical support and Ms. Ellen Hagen, Ms. Sandra Berven, and Lisa Nichols, B.S., for preparation of the manuscript.
4. 5.
6.
7.
8.
REFERENCES 1. Malter HE, Cohen J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res 1989;24:67-80. 2. Malter HE, Cohen J. Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 1989;51:13948. 3. Cordon JW, Talansky BE. Assisted fertilization by zona drill-
994
Hu et al. Nonselective assisted hatching
9.
10.
ing: a mouse model for correction of oligospermia. J Exp Zool 1986; 239:347-54. Cohen J. Assisted hatching of human embryos. J In Vitro Fert Embryo Transf 1991;8:179-90. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer M, 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. 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. Wiemer RK, Hu Y, Cuervo M, Genetis P, Leibowitz D. The combination of coculture and selective assisted hatching: results from their clinical application. Fertil Steril 1994;61: 105-10. Droesch K, Muasher SJ, Brzyski RG, Jones GS, Simonetti S, Liu H-C, et al. Value of suppression with a gonadotropin stimulation for in vitro fertilization. Fertil Steril 1989;51: 292-7. Garcia E, Padilla SL, Bayati J, Baramki TA. Follicular phase gonadotropin-releasing hormone agonist and human gonadotropins: a better alternative for ovulation induction in in vitro fertilization. Fertil Steril 1990;53:302-5. Quinn P, Kerin F, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493-8.
Fertility and Sterility®