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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
FERTILITYAND STERILITY@
Vol. 67, No. 6, May 1997
Copyright o 1997AmericanSocietyforReproductive Medicine
Printed on mid-free paper in U. S. A.
Published by Elsevier Science Inc.
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
Kuang-Han Chao, M.D. Shee-Uan Chen, M.D. Hsin-Fu Chen, M.D. Department Republic
Ming-Yih Wu, M.D. Yu-Shih Yang, M.D., Ph.D. Hong-Nerng Ho, M.D.*
of Obstetrics and Gynecology,
College of Medicine
and the Hospital, National
Taiwan
University,
Taipei, Taiwan,
of China
Objective: To assess the effect of augmenting IVF with assisted hatching in the treatment of patients with repeated M? failures. Design: Prospective randomized study. Setting: Division of Reproductive Endocrinology and Infertility of National Taiwan University Hospital. Patient(s): From July 1993 to February 1996, 49 patients with repeatedly failed IVF were treated with assisted hatching and were compared with 51 control subjects without assisted hatching. Intervention(s): Assisted hatching. Main Outcome Measure(s): Pregnancy rate and implantation rate per embryo after IVFET or IVF-tubal ET (TET) were measured. Result(s): The pregnancy rate (PR) in the assisted hatching group was found to be 36.7% compared with 19.6% in the control group, but the difference was not significant. When only patients receiving IVF-ET were considered, it was observed that the PR was significantly higher in the assisted hatching group than the control group (42.4% versus 16.1%). With IVF-TET however, the PR was found to be similar in both assisted hatching and control groups (25.0% and 25.0%, respectively). The rate of embryonic implantation in the IVF-ET patients was ll.O%, which was significantly higher than that of control embryos (3.7%). Conclusion(s): These results implied that IVF-ET, combined with assisted hatching, may improve the PR and implantation rate in patients with repeated IVF failures, but the same was not true in the case of IVF-TET. (Fertil Steril@ 1997;67:904-8. 0 1997 by American Society for Reproductive Medicine.) Key Words: Assisted hatching, implantation rate, pregnancy rate, repeated in vitro fertilization failures
Since the advent of IVF, improved pregnancy rates (PRs) per ET or tubal ET (TET) have remained a constant but elusive goal. The two methodologies, coculture and assisted hatching of human embryos, claim a significant improvement in hatching and im-
plantation rate per embryo (l-3). Although much controversy has surrounded the efficacy of assisted hatching (41, three randomized, controlled trials demonstrated a clear benefit from assisted hatching, particularly in the case of poor-prognosis patients (5-7).
ReceivedNovember11, 1996; revised and accepted January 8, 1997. * Reprint requests: Hong-Nerng Ho, M.D., Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, 10016, Taipei, Taiwan, Republic of China (FAX: 886-2-341-8557).
904
Two particular findings have been crucial in deciding the implementation of assisted hatching clinically. First, it has been observed that cleaved embryos with a good prognosis for implantation have a reduced zona thickness, presumably, in preparation for subsequent hatching (8, 9). Second, microsurgi-
0015-0282/97/$17.00 PI1 SO0150282(97)00121-O
tally fertilized or biopsied embryos with artificial gaps in their zonae appear to have high rates of implantation (10, 11). Assisted hatching was first tested experimentally by introducing small incisions in the zonae of human four-cell embryos using a mechanical method (5). Our previous study using the mouse model showed that a significantly higher percentage of partial zona dissection (PZD) embryos than controls initiated and completed hatching (12). The purpose of this study was to test the efficiency of assisted hatching by comparing the results of standard IVF methodology to the same IVF protocol with the addition of assisted hatching. Because the efficacy of assisted hatching remains controversial and experimental, we focus only on tlie patients with repeated IVF failures whose fertilization rates were normal. MATERIALS
AND METHODS
Subjects
In the span between July 1993 and February 1996, 100 consecutive patients were identified as having multiple prior IVF cycle failures because of no implantation (at least two failures of M? with an adequate number of oocytes retrieved, normal fertilization and cleavage rate, and at least two embryos available for transfer). They were treated with further IVF programs. A total of 64 M?-ET patients and 36 IVF-TET patients were assigned to a control group and an assisted hatching group prospectively by a computer-generated randomization sequence. The mean age, mean levels of day 3 FSH and LH, and number of embryos transferred were all assessed. The protocol was approved by our internal ethical committee. Procedures of Ovarian Stimulation, Oocyte Retrieval, M?-ET or TET and Assisted Hatching
Ovarian stimulation was accomplished with FSH (Metrodin; Serono, Rome, Italy) and hMG (Pergonal; Serono) after pituitary down-regulation with GnRH analogue (buserelin acetate; Hoechst Laboratory, Frankfurt, Germany). The dosage of hMG was adjusted individually according to the ovarian response as previously described (13). When at least two leading follicles had reached a mean diameter of 218 mm and serum Ez level was appropriate, hCG (10,000 IU, Pregnyl; Organon, Oss, The Netherlands-) was administered intramuscularly (IM). Oocyte retrieval was scheduled to take place 34 to 36 hours later by transvaginal ultrasound guidance. Six hours later, the oocytes were inseminated and the pronucleate state was examined 16 to 18 hours after
Vol. 67, No. 5, May 1997
insemination. The normally fertilized oocytes were selected and cultured for one more day. Embryos at two- to four-cell stage of the control group were transferred to the uterus (ET) or to the fallopian tubes (TET) when at least one tube was patent. All embryos in the assisted hatching group were also cultured for 2 days. On the morning of the 2nd day of embryo culture (approximately 48 hours after oocyte retrieval), assisted hatching was performed using the PZD technique (5, 12). Briefly, micromanipulation was carried out in dishes containing 20PL droplets of human tubal fluid culture medium overlaid with pre-equilibrated warmed mineral oil. Embryos were stabilized by a holding pipette at the 9 o’clock position so that the indentation between two adjacent blastomeres was between the 2 o’clock and 3 o’clock positions. The zona pellucida (ZP) was then pierced with the microneedle on that side until the needle tip was visible in the perivitelline space. The ZP was moved upward and the microneedle was pushed tangentially through the space between the zona and blastomere until it pierced through the zona again. The suction was discontinued and the holding pipette rubbed over the small part of the zona trapped against the microneedle. The procedure was completed when the area between the two sides pierced by the microneedle was opened, leaving a narrow incision about one sixth of the circumference of the zona. Embryos were rinsed several times and returned to standard culture conditions for an additional 4 to 6 hours before transfer. Embryo transfer was performed as atraumatically as possible into the uterine cavity (ET) or fallopian tube (TET) by laparoscope. The luteal phase was supplemented with 50 mg IM P in oil. The patients were followed by P-hCG and sonography. The pregnancy was confirmed by positive /?-hCG 14 days after transfer, and implantation was defined as presence of gestational sac in uterus by ultrasonography. Statistical Analysis
All results are expressed as mean _+SD. Statistical analysis was implemented with the Student’s ttest and x2 test. A P value less than 0.05 was considered statistically significant. RESULTS
The clinical characteristics of the assisted hatching and control groups by ET or TET are presented in Table 1. The age of patients in both assisted hatching and control groups was identical, respectively, in ET and TET patients. The serum levels of day 3 FSH and LH, total retrieved oocytes, and fertilization rate were not statistically different between as-
Chao
et al.
Assisted hatching in IVF-ET program
905
Table 1 The Clinical Characteristics of Patients in Four Different Groups ET
Total number of patients Maternal age (y)* Day 3 FSH (mIU/mL)t LH (mIU/mL)t Total eggs retrieved per patient Fertilization rate (%) No. of total embryos per patient No. of total embryos transferred Number of blastomeres
Control
Assisted hatching
31 34.0 5 3.9
33 36.5 ‘- 5.2
36.4:
6.9 4.0 8.3 75.1 5.7 4.3 3.9
6.7 4.3 8.2 76.0 5.8 4.7 4.1
7.3 4.3 7.1 73.4 5.1 4.1 3.5
2 2.9 ? 3.2 4 5.1 4 24.4 ? 3.9 Z 2.0 t 1.6
sisted hatching and control groups for the groups as a whole. The mean number of embryos transferred was 4.3 versus 4.7 in ET patients and 4.1 versus 3.9 in TET patients, respectively, and all had no significant difference. The number of blastomeres in the two populations also did not reveal significant differences. Table 2 summarizes the PRs and implantation rates of the assisted hatching and control groups. The PR was significantly higher in the assisted hatching group than that in the control group (42.4% versus 16.1%, P = 0.0211 when only ET patients were considered. Nevertheless, there was no difference when all the patients were included (ET and TET patients). Similarly, implantation rates (gestational sac per embryo replaced) in ET patients increased significantly (P = 0.021) from 3.7% in the control group to 11.0% in the assisted hatching group. This result suggested that PZD of embryos may be advantageous for embryo implantation. The same beneficial effect on implantation could not be demonstrated in those patients receiving IVF-TET. The implantation rate in total assisted hatching
Table 2 Results of In Vitro Fertilization in the Assisted Hatching and Control Groups* Control
Assisted hatching
P value
5/31 (16.1) 5120 (25.0) lo/51 (19.6)
14/33 (42.4) 4/16 (25.0) 18149 (36.7)
0.021 1.000 0.057
17/155 (11.0) 4/62 (6.5) 2l/217 (9.7)
0.021 0.611 0.112
51134 (3.7) 7/80 (8.8) 12/214 (5.6)
* Values in parentheses are percentages.
906
i 2.4 + 2.8 ? 4.7 ? 21.8 ? 3.1 t 2.0 k 1.5
Control 4.1 2 2.8 5 3.1 -+ 4.3 ” 16.6 2 3.3 2 1.4 -+ 1.0
Assisted hatching 16 36.8 ? 4.3 8.7 3.7 6.8 64.4 4.0 3.9 3.8
2 3.9 r 2.1 ? 3.3 2 22.6 + 1.6 2 1.7 k 1.3
‘FConversion factor to SI unit, 1.00.
* Values are means ? SD.
Pregnancy rate IVF-ET IVF-TET Total Implantation rate (gestational sac per embryo) IVF-ET IVF-TET Total
TET
Chao et al. Assisted hatching in IVF-ET program
group was higher than that in total control group (9.7% versus 5.6%), but the difference was not significant (P = 0.112). There was no difference in the abortion rate and the incidence of congenital anomaly when the pregnancy outcome was considered. DISCUSSION A number of procedures have been considered in recent years to enhance embryonic implantation after IVF. Assisted hatching, or artificial opening of the ZP, has been reported with promising results (5, 6, 14); however, routine application was not implemented mainly for three reasons. First, some of the spare embryos that were observed for prolonged periods became trapped in the narrow micromanipulation openings with relatively thick zona during hatching (15, 161. These observations were confirmed in a mouse model (17). Second, there was a possibility of other embryos being damaged during embryo replacement prior to the formation of structural junctions between the blastomeres to cause the loss of blastomeres (14, 18). Third, it also deprived the embryo of its protective coat, which protected against any detrimental factors in the female reproductive tract (19). In vitro fertilization patients with prior IVF failures have been shown to have a poor prognosis for pregnancy (20). The result of this study indicates that PRs can be enhanced by augmenting IVF with assisted hatching, presumably because poor-prognosis embryos can be rescued by assisted hatching. It has been proposed that advanced ovarian age is related to abnormal zona synthesis (211. The exact mechanism by which assisted hatching enhances implantation, however, is yet to be proved, although at least three possibilities exist. First, in prior IVF failure patients, “zona hardening” (22) may originate from suboptimal in vitro cul-
Fertility and Sterility@
ture conditions, although the prevalence of this physical characteristic in these patients remains to be demonstrated. It might relate to physical alteration of the zona, including thickness, sensitivity to enzymes, zona deformation after suction and breakdown of the matrix by acidic dissolution. In these cases, assisted hatching simply overcomes a mechanical barrier from the hardened zona, allowing implantation to occur. Second, the cellular energy requirement for hatching may be insufficient in patients exhibiting repeated IVF failure. Assisted hatching may decrease the energy required by an embryo to complete the hatching process (20). Third, relative failure to implant may be because of asynchrony between the embryo and the endometrial implantation window among patients with ovarian stimulation. Precise synchronization between embryo and endometrium is essential for implantation. Normal dissolution of the zona (hatching) occurs about 120 hours (5 days) after fertilization, whereas implantation in the normal (unstimulated) cycle seems to begin about 120 hours after fertilization (23). Among stimulated patients, the window of implantation has been found to be moved forward to between 72 and 120 hours after fertilization. Thus, if hatching occurs at 120 hours, there exists a timelimited opportunity for implantation. Assisted hatching seems to allow earlier hatching (15) and implantation (7) and thus obviates the closing of the implantation window in stimulated patients. On average, implantation was observed to occur approximately 1 day earlier in the assisted hatching group than in controls (7). The fallopian tubes may produce some growth factors to promote the embryo development and lysin production (241, so that assisted hatching may not be beneficial to the TET patients. It might even be deleterious because the contraction of the fallopian tubes may cause the embryos to hatch prematurely (14) and the embryos may be damaged because of compression during passage through the oviduct (25). Our results indicate that assisted hatching by PZD for patients with a history of previous failed IVF is advantageous in ET, but not in TET. Therefore, application of assisted hatching to the general IVF population is not advised. The current results recently have led us to implement selective assisted hatching by PZD routinely in consenting IVF-ET patients with repeated failure of prior IVF, but not in TET patients. Further evaluation is needed to understand whether there are any growth factors in the fallopian tubes that tend to enhance the embryo hatching in TET patients. Moreover, the criteria for application in individual embryos also require further clarification.
Vol. 67, No. 5, May 1997
Acknowledgment. We gratefully acknowledged Mrs. Jane-Ru Lin, the technician of the laboratory of reproductive endocrinology and infertility, National Taiwan University Hospital, for her technical assistance in this study.
REFERENCES
1. Bongso A, Ng SC, Fong CY, Anandakumar C, Marshall B, Edirisinghe R, et al. Improved pregnancy rate after transfer of embryos grown in human fallopian tubal cell coculture. Fertil Steril 1992;58:569-74. 2. Khalifa EA, Tucker MJ, Hunt PP, Hamidi J. Improved hatching in mouse embryos brought about by combined partial zona dissection and co-culture. Hum Reprod 1993;8:599663. 3. Tucker MJ, Ingargiola PE, Massey JB, Morton PC, Wiemer KE, Wiker SR, et al. Assisted hatching with or without bovine oviductal epithelial cell co-culture for poor prognosis in-vitro fertilization patients. Hum Reprod 1994;9:1528-31. 4. Tucker MJ, Luecke NM, Wiker SR, Wright G. Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate. J Assist Reprod Genet 1993; 10:187-91. 5. 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;5:713. 6. 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. 7. Liu HC, Cohen J, Alikani M, Noyes N, Rosenwaks Z. Assisted hatching facilitates earlier implantation. Fertil Steril 1993; 60:871-5. a. Cohen J, Inge KL, Suzman M, Wiker S, Wright G. Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil Steril 1989;51:820-7. 9. Wright G, Wiker S, Elsner C, Kort H, Massey J, Mitchell D, et al. Observations on the morphology of human zygotes, pronuclei and nucleoli and implications for cryopreservation. Hum Reprod 1990;5:109-15. 10. Cohen J, Alikani M, Malter HE, Adler A, Talansky BE, Rosenwaks Z. Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology. Fertil Steril 1991; 56:696-706. 11. Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344: 768-70. 12. Chen SU, Ho HN, Chen HF, Chao KH, Wu MY, Huang SC, et al. Effect of assisted hatching by partial zona dissection on mouse embryos in vitro. J Formos Med Assoc 1995;94:463-8. 13. Ho HN, Yang YS, Hsieh RP, Lin HR, Chen SU, Chen HF, et al. Sharing of human leukocyte antigens (HLA) in couples with unexplained infertility affects the success of in vitro fertilization and tubal embryo transfer. Am J Obstet Gynecol 1994;170:63-71. 14. Cohen J. Assisted hatching of human embryos. J In Vitro Fert Embryo Transf 1991;8:179-90. 15. Malter H, Cohen J. Blastocyst formation and hatching in vitro following zona drilling in mouse and human embryos. Gamete Res 1989;24:67-80.
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Assisted
hatching in IVF-ET program
907
16. 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. 17. Cohen J, Feldberg D. Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo. Mol Reprod Dev 1991;30:70-8. 18. Dale B, Gualitieri R, Talevi R, Tosti E, Santella L, Elder K. Intercellular communication in the early human embryo. Mol Reprod Dev 1991;29:22-8. 19. Cohen J, Malter H, Elmer C, Kort H, Massey J, Mayer MP. Immunosuppression supports implantation of zona pellucida dissected human embryos. Fertil Steril 1990;53:662-5. 20. Schoolcraft WB, Schlenker T, Gee M, Jones GS, Jones HW. Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril 1994;62:551-4. 21. Drobnis EZ, Andrew JB, Katz DF. Biophysical properties of
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Assisted hatching in IVF-ETprogram
22.
23.
24.
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the zona pellucida measured by capillary suction: is zona hardening a mechanical phenomenon? J Exp Zoo1 1988; 245206-19. 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. Psychoyos A. De Lataste a la “fenetre d’implantation”: cent ans de decouvertes fascinantes. [From Lataste to the “window of implantation”: 100 years of fascinating discoveries.] Contraception, Fertilite, Sexualiti 1993;21:333-8. Gordon JW, Dapunt U. A new mouse model for embryos with a hatching deficiency and its use to elucidate the mechanism of blastocyst hatching. Fertil Steril 1993;59:1296-301. Nichols J, Gardner RL. Effect of damage to the zona pellucida on development of preimplantation embryos in the mouse. Hum Reprod 1989; 4: 180-7.
Fertility- and Ste&ty@
Vol. 67, No. 6, May 1997
Copyright o 1997AmericanSocietyforReproductive Medicine
Printed on mid-free paper in U. S. A.
Published by Elsevier Science Inc.
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
Kuang-Han Chao, M.D. Shee-Uan Chen, M.D. Hsin-Fu Chen, M.D. Department Republic
Ming-Yih Wu, M.D. Yu-Shih Yang, M.D., Ph.D. Hong-Nerng Ho, M.D.*
of Obstetrics and Gynecology,
College of Medicine
and the Hospital, National
Taiwan
University,
Taipei, Taiwan,
of China
Objective: To assess the effect of augmenting IVF with assisted hatching in the treatment of patients with repeated M? failures. Design: Prospective randomized study. Setting: Division of Reproductive Endocrinology and Infertility of National Taiwan University Hospital. Patient(s): From July 1993 to February 1996, 49 patients with repeatedly failed IVF were treated with assisted hatching and were compared with 51 control subjects without assisted hatching. Intervention(s): Assisted hatching. Main Outcome Measure(s): Pregnancy rate and implantation rate per embryo after IVFET or IVF-tubal ET (TET) were measured. Result(s): The pregnancy rate (PR) in the assisted hatching group was found to be 36.7% compared with 19.6% in the control group, but the difference was not significant. When only patients receiving IVF-ET were considered, it was observed that the PR was significantly higher in the assisted hatching group than the control group (42.4% versus 16.1%). With IVF-TET however, the PR was found to be similar in both assisted hatching and control groups (25.0% and 25.0%, respectively). The rate of embryonic implantation in the IVF-ET patients was ll.O%, which was significantly higher than that of control embryos (3.7%). Conclusion(s): These results implied that IVF-ET, combined with assisted hatching, may improve the PR and implantation rate in patients with repeated IVF failures, but the same was not true in the case of IVF-TET. (Fertil Steril@ 1997;67:904-8. 0 1997 by American Society for Reproductive Medicine.) Key Words: Assisted hatching, implantation rate, pregnancy rate, repeated in vitro fertilization failures
Since the advent of IVF, improved pregnancy rates (PRs) per ET or tubal ET (TET) have remained a constant but elusive goal. The two methodologies, coculture and assisted hatching of human embryos, claim a significant improvement in hatching and im-
plantation rate per embryo (l-3). Although much controversy has surrounded the efficacy of assisted hatching (41, three randomized, controlled trials demonstrated a clear benefit from assisted hatching, particularly in the case of poor-prognosis patients (5-7).
ReceivedNovember11, 1996; revised and accepted January 8, 1997. * Reprint requests: Hong-Nerng Ho, M.D., Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, 10016, Taipei, Taiwan, Republic of China (FAX: 886-2-341-8557).
904
Two particular findings have been crucial in deciding the implementation of assisted hatching clinically. First, it has been observed that cleaved embryos with a good prognosis for implantation have a reduced zona thickness, presumably, in preparation for subsequent hatching (8, 9). Second, microsurgi-
0015-0282/97/$17.00 PI1 SO0150282(97)00121-O
tally fertilized or biopsied embryos with artificial gaps in their zonae appear to have high rates of implantation (10, 11). Assisted hatching was first tested experimentally by introducing small incisions in the zonae of human four-cell embryos using a mechanical method (5). Our previous study using the mouse model showed that a significantly higher percentage of partial zona dissection (PZD) embryos than controls initiated and completed hatching (12). The purpose of this study was to test the efficiency of assisted hatching by comparing the results of standard IVF methodology to the same IVF protocol with the addition of assisted hatching. Because the efficacy of assisted hatching remains controversial and experimental, we focus only on tlie patients with repeated IVF failures whose fertilization rates were normal. MATERIALS
AND METHODS
Subjects
In the span between July 1993 and February 1996, 100 consecutive patients were identified as having multiple prior IVF cycle failures because of no implantation (at least two failures of M? with an adequate number of oocytes retrieved, normal fertilization and cleavage rate, and at least two embryos available for transfer). They were treated with further IVF programs. A total of 64 M?-ET patients and 36 IVF-TET patients were assigned to a control group and an assisted hatching group prospectively by a computer-generated randomization sequence. The mean age, mean levels of day 3 FSH and LH, and number of embryos transferred were all assessed. The protocol was approved by our internal ethical committee. Procedures of Ovarian Stimulation, Oocyte Retrieval, M?-ET or TET and Assisted Hatching
Ovarian stimulation was accomplished with FSH (Metrodin; Serono, Rome, Italy) and hMG (Pergonal; Serono) after pituitary down-regulation with GnRH analogue (buserelin acetate; Hoechst Laboratory, Frankfurt, Germany). The dosage of hMG was adjusted individually according to the ovarian response as previously described (13). When at least two leading follicles had reached a mean diameter of 218 mm and serum Ez level was appropriate, hCG (10,000 IU, Pregnyl; Organon, Oss, The Netherlands-) was administered intramuscularly (IM). Oocyte retrieval was scheduled to take place 34 to 36 hours later by transvaginal ultrasound guidance. Six hours later, the oocytes were inseminated and the pronucleate state was examined 16 to 18 hours after
Vol. 67, No. 5, May 1997
insemination. The normally fertilized oocytes were selected and cultured for one more day. Embryos at two- to four-cell stage of the control group were transferred to the uterus (ET) or to the fallopian tubes (TET) when at least one tube was patent. All embryos in the assisted hatching group were also cultured for 2 days. On the morning of the 2nd day of embryo culture (approximately 48 hours after oocyte retrieval), assisted hatching was performed using the PZD technique (5, 12). Briefly, micromanipulation was carried out in dishes containing 20PL droplets of human tubal fluid culture medium overlaid with pre-equilibrated warmed mineral oil. Embryos were stabilized by a holding pipette at the 9 o’clock position so that the indentation between two adjacent blastomeres was between the 2 o’clock and 3 o’clock positions. The zona pellucida (ZP) was then pierced with the microneedle on that side until the needle tip was visible in the perivitelline space. The ZP was moved upward and the microneedle was pushed tangentially through the space between the zona and blastomere until it pierced through the zona again. The suction was discontinued and the holding pipette rubbed over the small part of the zona trapped against the microneedle. The procedure was completed when the area between the two sides pierced by the microneedle was opened, leaving a narrow incision about one sixth of the circumference of the zona. Embryos were rinsed several times and returned to standard culture conditions for an additional 4 to 6 hours before transfer. Embryo transfer was performed as atraumatically as possible into the uterine cavity (ET) or fallopian tube (TET) by laparoscope. The luteal phase was supplemented with 50 mg IM P in oil. The patients were followed by P-hCG and sonography. The pregnancy was confirmed by positive /?-hCG 14 days after transfer, and implantation was defined as presence of gestational sac in uterus by ultrasonography. Statistical Analysis
All results are expressed as mean _+SD. Statistical analysis was implemented with the Student’s ttest and x2 test. A P value less than 0.05 was considered statistically significant. RESULTS
The clinical characteristics of the assisted hatching and control groups by ET or TET are presented in Table 1. The age of patients in both assisted hatching and control groups was identical, respectively, in ET and TET patients. The serum levels of day 3 FSH and LH, total retrieved oocytes, and fertilization rate were not statistically different between as-
Chao
et al.
Assisted hatching in IVF-ET program
905
Table 1 The Clinical Characteristics of Patients in Four Different Groups ET
Total number of patients Maternal age (y)* Day 3 FSH (mIU/mL)t LH (mIU/mL)t Total eggs retrieved per patient Fertilization rate (%) No. of total embryos per patient No. of total embryos transferred Number of blastomeres
Control
Assisted hatching
31 34.0 5 3.9
33 36.5 ‘- 5.2
36.4:
6.9 4.0 8.3 75.1 5.7 4.3 3.9
6.7 4.3 8.2 76.0 5.8 4.7 4.1
7.3 4.3 7.1 73.4 5.1 4.1 3.5
2 2.9 ? 3.2 4 5.1 4 24.4 ? 3.9 Z 2.0 t 1.6
sisted hatching and control groups for the groups as a whole. The mean number of embryos transferred was 4.3 versus 4.7 in ET patients and 4.1 versus 3.9 in TET patients, respectively, and all had no significant difference. The number of blastomeres in the two populations also did not reveal significant differences. Table 2 summarizes the PRs and implantation rates of the assisted hatching and control groups. The PR was significantly higher in the assisted hatching group than that in the control group (42.4% versus 16.1%, P = 0.0211 when only ET patients were considered. Nevertheless, there was no difference when all the patients were included (ET and TET patients). Similarly, implantation rates (gestational sac per embryo replaced) in ET patients increased significantly (P = 0.021) from 3.7% in the control group to 11.0% in the assisted hatching group. This result suggested that PZD of embryos may be advantageous for embryo implantation. The same beneficial effect on implantation could not be demonstrated in those patients receiving IVF-TET. The implantation rate in total assisted hatching
Table 2 Results of In Vitro Fertilization in the Assisted Hatching and Control Groups* Control
Assisted hatching
P value
5/31 (16.1) 5120 (25.0) lo/51 (19.6)
14/33 (42.4) 4/16 (25.0) 18149 (36.7)
0.021 1.000 0.057
17/155 (11.0) 4/62 (6.5) 2l/217 (9.7)
0.021 0.611 0.112
51134 (3.7) 7/80 (8.8) 12/214 (5.6)
* Values in parentheses are percentages.
906
i 2.4 + 2.8 ? 4.7 ? 21.8 ? 3.1 t 2.0 k 1.5
Control 4.1 2 2.8 5 3.1 -+ 4.3 ” 16.6 2 3.3 2 1.4 -+ 1.0
Assisted hatching 16 36.8 ? 4.3 8.7 3.7 6.8 64.4 4.0 3.9 3.8
2 3.9 r 2.1 ? 3.3 2 22.6 + 1.6 2 1.7 k 1.3
‘FConversion factor to SI unit, 1.00.
* Values are means ? SD.
Pregnancy rate IVF-ET IVF-TET Total Implantation rate (gestational sac per embryo) IVF-ET IVF-TET Total
TET
Chao et al. Assisted hatching in IVF-ET program
group was higher than that in total control group (9.7% versus 5.6%), but the difference was not significant (P = 0.112). There was no difference in the abortion rate and the incidence of congenital anomaly when the pregnancy outcome was considered. DISCUSSION A number of procedures have been considered in recent years to enhance embryonic implantation after IVF. Assisted hatching, or artificial opening of the ZP, has been reported with promising results (5, 6, 14); however, routine application was not implemented mainly for three reasons. First, some of the spare embryos that were observed for prolonged periods became trapped in the narrow micromanipulation openings with relatively thick zona during hatching (15, 161. These observations were confirmed in a mouse model (17). Second, there was a possibility of other embryos being damaged during embryo replacement prior to the formation of structural junctions between the blastomeres to cause the loss of blastomeres (14, 18). Third, it also deprived the embryo of its protective coat, which protected against any detrimental factors in the female reproductive tract (19). In vitro fertilization patients with prior IVF failures have been shown to have a poor prognosis for pregnancy (20). The result of this study indicates that PRs can be enhanced by augmenting IVF with assisted hatching, presumably because poor-prognosis embryos can be rescued by assisted hatching. It has been proposed that advanced ovarian age is related to abnormal zona synthesis (211. The exact mechanism by which assisted hatching enhances implantation, however, is yet to be proved, although at least three possibilities exist. First, in prior IVF failure patients, “zona hardening” (22) may originate from suboptimal in vitro cul-
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ture conditions, although the prevalence of this physical characteristic in these patients remains to be demonstrated. It might relate to physical alteration of the zona, including thickness, sensitivity to enzymes, zona deformation after suction and breakdown of the matrix by acidic dissolution. In these cases, assisted hatching simply overcomes a mechanical barrier from the hardened zona, allowing implantation to occur. Second, the cellular energy requirement for hatching may be insufficient in patients exhibiting repeated IVF failure. Assisted hatching may decrease the energy required by an embryo to complete the hatching process (20). Third, relative failure to implant may be because of asynchrony between the embryo and the endometrial implantation window among patients with ovarian stimulation. Precise synchronization between embryo and endometrium is essential for implantation. Normal dissolution of the zona (hatching) occurs about 120 hours (5 days) after fertilization, whereas implantation in the normal (unstimulated) cycle seems to begin about 120 hours after fertilization (23). Among stimulated patients, the window of implantation has been found to be moved forward to between 72 and 120 hours after fertilization. Thus, if hatching occurs at 120 hours, there exists a timelimited opportunity for implantation. Assisted hatching seems to allow earlier hatching (15) and implantation (7) and thus obviates the closing of the implantation window in stimulated patients. On average, implantation was observed to occur approximately 1 day earlier in the assisted hatching group than in controls (7). The fallopian tubes may produce some growth factors to promote the embryo development and lysin production (241, so that assisted hatching may not be beneficial to the TET patients. It might even be deleterious because the contraction of the fallopian tubes may cause the embryos to hatch prematurely (14) and the embryos may be damaged because of compression during passage through the oviduct (25). Our results indicate that assisted hatching by PZD for patients with a history of previous failed IVF is advantageous in ET, but not in TET. Therefore, application of assisted hatching to the general IVF population is not advised. The current results recently have led us to implement selective assisted hatching by PZD routinely in consenting IVF-ET patients with repeated failure of prior IVF, but not in TET patients. Further evaluation is needed to understand whether there are any growth factors in the fallopian tubes that tend to enhance the embryo hatching in TET patients. Moreover, the criteria for application in individual embryos also require further clarification.
Vol. 67, No. 5, May 1997
Acknowledgment. We gratefully acknowledged Mrs. Jane-Ru Lin, the technician of the laboratory of reproductive endocrinology and infertility, National Taiwan University Hospital, for her technical assistance in this study.
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