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Should all embryos from day 1 rescue intracytoplasmic sperm injection be transferred during frozen–thawed cycles?
Should all embryos from day 1 rescue intracytoplasmic sperm injection be transferred during frozen–thawed cycles? A retrospective analysis of a 5-year rescue intracytoplasmic sperm injection (ICSI) experience performed in 17 cases of complete IVF fertilization failure showed that the ongoing pregnancy rate per embryo transfer (ET) is much higher in a subsequent frozen–thawed cycles (40%, 2/5) than following a fresh ET (6.7%, 1/15). These results suggest that cryopreservation of all embryos, subsequently used in frozen–thawed cycles, might improve rescue ICSI outcome throughout a better synchronization of the embryo development with the endometrium. (Fertil Steril 2010;94:1157–8. 2010 by American Society for Reproductive Medicine.) Key Words: Fertilization failure, rescue ICSI, cryopreservation, synchronization, endometrium Unexpected complete fertilization failure in case of previously proven female infertility occurs in about 5% to 10% of conventional in vitro fertilization (cIVF) cycles (1). Sperm defect, oocytes’ abnormality, and disturbance in sperm–oocyte interaction may account for this infrequent situation. To overcome fertilization failure, some investigators have suggested to perform a day 1 intracytoplasmic sperm injection (ICSI) of unfertilized mature oocytes, the so-called ‘‘rescue’’ ICSI (2–5). Although some pregnancies and live births have been reported following fresh (5–7) or frozen–thawed (3, 5) embryo transfers (ET), this procedure is still controversial because of a high incidence of genetic abnormalities in the resulting embryos (7–9) and of a poor outcome (2, 10). Nathalie Sermondade, M.D.a Jean-No€el Hugues, M.D., Ph.D.b Isabelle Cedrin-Durnerin, M.D., Ph.D.b Christophe Poncelet, M.D., Ph.D.c Brigitte Benzacken, M.D., Ph.D.a Rachel Levy, M.D., Ph.D.a Christophe Sifer, M.D.a a Service d’Histologie-Embryologie-Cytog en etique-Biologie de la Reproduction-CECOS, Centre Hospitalier Universitaire Jean Verdier, Assistance Publique–H^ opitaux de Paris, Bondy, France b Service de M edecine de la Reproduction, Centre Hospitalier Universitaire Jean Verdier, Assistance Publique–H^ opitaux de Paris, Bondy, France c Service de Gyn ecologie-Obst etrique, Centre Hospitalier Universitaire Jean Verdier, Assistance Publique–H^ opitaux de Paris, Bondy, France Received September 22, 2009; revised November 16, 2009; accepted December 1, 2009; published online January 15, 2010. N.S. has nothing to disclose. J-N.H. has nothing to disclose. I.C-D. has nothing to disclose. C.P. has nothing to disclose. B.B. has nothing to disclose. R.L. has nothing to disclose. C.S. has nothing to disclose. Reprint requests: Christophe Sifer, M.D., Service d’Histologie-Embryone tique-Biologie de la Reproduction, Centre Hospitalier logie-Cytoge ^ pitaux de Paris, Universitaire Jean Verdier, Assistance Publique–Ho 93140 Bondy, France (FAX: þ33 1 48 02 68 79; E-mail: christophe. [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2009.12.001
The low implantation rate could be explained either by the in vitro aging of cultured oocytes before ICSI or by the asynchrony between the embryo development stage and the endometrial secretory pattern. To address this issue, we retrospectively reviewed a 5-year experience of rescue ICSI and analyzed the cycle outcome following transfer of fresh and frozen embryos. This retrospective study analyzed cases of unexplained failure of fertilization after cIVF observed in our infertility center between May 2004 and July 2009. Ovarian stimulations and cumulus–oocytes complexes retrieval were performed by standard procedures as previously described (11). Cumulus–oocytes complexes were routinely inseminated 3 to 5 hours after pickup with 10,000 motile selected spermatozoa in 30-mL appropriate embryo culture medium microdroplets under mineral oil and examined for evidence of fertilization 18 hours after insemination. Oocytes were considered normally fertilized when two pronuclei (2 PN) and two polar bodies were observed. The diploid fertilization rate was defined by the ratio of 2 PN and metaphase two oocytes. Metaphase two oocytes were considered as unfertilized when no pronucleus and only one polar body were observed into the ooplasm. In the case of complete cIVF fertilization failure, ICSI of unfertilized mature oocytes was performed as previously described (12) on day 1 with sperm from the original day 0 insemination. All included patients signed an informed consent form before rescue ICSI procedure. Oocytes were examined for evidence of fertilization 18 hours after ICSI and embryo quality was evaluated 48/72 hours after ICSI, according to previously described criteria (13). Good-quality embryos were transferred after enzymatic assisted hatching (AH) on day 3 after rescue ICSI. Good-quality supernumerary embryos were cryopreserved with a slow freezing protocol and transferred after AH in a subsequent thawing cycle. A clinical pregnancy was assessed by a positive fetal heartbeat on transvaginal ultrasound at 5 to 6 weeks following ET. Ongoing pregnancy was defined by an intrauterine pregnancy older than 12 weeks of amenorrhea. Data are presented as mean SD. Seventeen couples who experienced complete fertilization failure after cIVF provided written consent for a rescue ICSI procedure after receiving detailed information. Couple infertility included tubal factor (n ¼ 6), severe endometriosis (n ¼ 4),
Fertility and Sterility Vol. 94, No. 3, August 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
1157
ovulatory dysfunction (n ¼ 1), or idiopathic infertility (n ¼ 6). Women were 27 to 42 years old (35.5 3.6 years). Infertility duration was of 4.7 2.4 years. Basal blood FSH and antral follicle count determined at day 3 of a natural cycle was 7.5 2.1 IU/mL and 13.55 7.4, respectively. Controlled ovarian stimulation (COS) was performed using either a standard long GnRH agonist (n ¼ 8) or a GnRH antagonist (n ¼ 9) protocols. The total amount of rFSH administered was 2,219 1,069 IU and the duration of COS was 12.3 3.5 days. A mean of 7.47 3.53 unfertilized mature oocytes were microinjected. The 2 PN fertilization rate was 60.9%, whereas abnormal (R3 PN) fertilization rate was observed in 7.8% of injected oocytes. The percentage of good-quality embryo at the time of ET was of 70.6%. Fifteen fresh ETs of 2.35 1.17 embryos and 6 cryopreservations of 3.33 1.37 good-quality embryos were performed. After fresh ET, 1 ongoing pregnancy was obtained (pregnancy rate [PR] per ET ¼ 6.7%, per rescue ICSI cycle ¼ 5.9%). After embryo thawing (n ¼ 5 cycles), the survival rate was 85.7%, leading to five ETs of 2.40 0.55 embryos. Two ongoing pregnancies were obtained (PR per ET ¼ 40%, per thawing cycle ¼ 40%). One pregnancy led to the live birth after vaginal delivery of a healthy baby girl of 3,900 g weight at 39 weeks of gestation without complications. The other pregnancy is still ongoing after 20 weeks of gestation. This retrospective analysis of rescue ICSI shows that the PR is much higher following transfer of frozen–thawed good-quality embryos than after transfer of fresh embryos. The fertilization rate following ICSI of 1-day-old oocytes did not differ from other publications (5). However, the 3 PN fertilization rate after rescue ICSI was actually increased by 3.25-fold compared with that observed when oocytes are injected at day 0 (personnel data from 2 years consecutive ICSI results; 7.8% vs. 2.4%; P¼.0003). This result confirms previous reports (6–7, 10), and may be related to an abnormal ageing of oocytes in culture. In our study, a poor clinical outcome was observed following rescue ICSI procedure and
fresh ET. These data are in good agreement with previous studies reporting a large range of clinical PR (6.9% to 20.7%) (5–7) or even negative outcome (2, 10). In vitro aging of the oocyte could be responsible for this disappointing outcome after rescue ICSI (2). Indeed, a dramatic increase in cytogenetic abnormalities has been reported when the time interval between oocyte retrieval and fertilization is extended (9, 14). An alternative factor likely to contribute to the low PR following fresh ET get from rescue ICSI could be the asynchrony between embryo development and the endometrial secretory pattern. The endometrium is known to be receptive during a limited period of time, called the ‘‘implantation window’’ (15). Previous studies have shown that endometrial maturation according to Noyes’ criteria on the day of oocyte retrieval in IVF cycles is in advance by 2 to 4 days compared with natural cycles, regardless of controlled ovarian hyperstimulation protocols. In those studies, no ongoing clinical pregnancy was obtained if the endometrial advancement exceeds 3 days (16, 17). The procedure of day 1 rescue ICSI is likely to worsen this phenomenon by delaying the time of transfer. The promising ongoing PR observed in subsequent frozen–thawed cycles is in agreement with this assumption. Our results suggest that [1] the asynchrony between the embryo development and the endometrial receptivity could be the main contributing factor for low implantation rates observed after fresh ETs, and [2] that embryo cryopreservation with subsequent transfer in frozen–thawed cycles should be recommended to improve endometrial synchronization. In conclusion, day 1 rescue ICSI followed by embryo freezing could be a helpful tool to prevent from complete fertilization failure after cIVF and achieve live birth. Although prospective studies are needed to confirm these data, a systematic cryopreservation of all good-quality embryos obtained after rescue ICSI of unfertilized oocytes in cIVF could be an interesting option to better synchronize embryo development and endometrial receptivity.
REFERENCES 1. Mahutte NG, Arici A. Failed fertilization: is it predictable? Curr Opin Obstet Gynecol 2003;15:211–8. 2. Kuczynski W, Dhont M, Grygoruk C, Pietrewicz P, Redzko S, Szamatowicz M. Rescue ICSI of unfertilized oocytes after IVF. Hum Reprod 2002;17: 2423–7. 3. Lombardi E, Tiveron M, Inza R, Valcarcel A, Young E, Bisioli C. Live birth and normal 1-year follow-up of a baby born after transfer of cryopreserved embryos from rescue intracytoplasmic sperm injection of 1-day-old oocytes. Fertil Steril 2003;80:646–8. 4. Nagy ZP, Joris H, Liu J, Staessen C, Devroey P, Van Steirteghem AC. Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes. Hum Reprod 1993;8:2180–4. 5. Yuzpe AA, Liu Z, Fluker MR. Rescue intracytoplasmic sperm injection (ICSI)-salvaging in vitro fertilization (IVF) cycles after total or near-total fertilization failure. Fertil Steril 2000;73:1115–9. 6. Lundin K, Sjogren A, Hamberger L. Reinsemination of one-day-old oocytes by use of intracytoplasmic sperm injection. Fertil Steril 1996;66:118–21. 7. Morton PC, Yoder CS, Tucker MJ, Wright G, Brockman WD, Kort HI. Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes
1158
Sermondade et al.
8.
9.
10.
11.
12.
13.
after complete conventional fertilization failure. Fertil Steril 1997;68:488–91. Nagy Z, Staessen C, Liu J, Joris H, Devroey P, Van Steirteghem A. Prospective, auto-controlled study on reinsemination of failed-fertilized oocytes by intracytoplasmic sperm injection. Fertil Steril 1995;64:1130–5. Pehlivan T, Rubio C, Ruiz A, Navarro J, Remohi J, Pellicer A, et al. Embryonic chromosomal abnormalities obtained after rescue intracytoplasmic sperm injection of 1-day-old unfertilized oocytes. J Assist Reprod Genet 2004;21:55–7. Chen C, Kattera S. Rescue ICSI of oocytes that failed to extrude the second polar body 6 h post-insemination in conventional IVF. Hum Reprod 2003;18:2118–21. Huirne JA, Hugues JN, Pirard C, Fischl F, Sage JC, Pouly JL, et al. Cetrorelix in an oral contraceptivepretreated stimulation cycle compared with buserelin in IVF/ICSI patients treated with r-hFSH: a randomized, multicentre, phase IIIb study. Hum Reprod 2006;21:1408–15. Palermo G, Joris H, Devroey P, Van Steirteghem A. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17–8. Leniaud L, Poncelet C, Porcher R, Martin-Pont B, Cedrin-Durnerin I, Hugues J, et al. [Prospective
Rescue ICSI and embryo cryopreservation
14.
15.
16.
17.
evaluation of elective single-embryo transfer versus double-embryo transfer following in vitro fertilization: a two-year French hospital experience]. Gynecol Obstet Fertil 2008;36:159–65. DeUgarte CM, Li M, Jordan B, Hill D, DeCherney A and Surrey M. Rescue intracytoplasmic sperm injection and preimplantation genetic diagnosis in combination can result in pregnancy. Fertil Steril 2006;86: 200–2. Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 1999;340:1796–9. Kolibianakis E, Bourgain C, Albano C, Osmanagaoglu K, Smitz J, Van Steirteghem A, et al. Effect of ovarian stimulation with recombinant follicle-stimulating hormone, gonadotropin releasing hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil Steril 2002;78:1025–9. Ubaldi F, Bourgain C, Tournaye H, Smitz J, Van Steirteghem A, Devroey P. Endometrial evaluation by aspiration biopsy on the day of oocyte retrieval in the embryo transfer cycles in patients with serum progesterone rise during the follicular phase. Fertil Steril 1997;67:521–6.
Vol. 94, No. 3, August 2010
0015-0282/$36.00 doi:10.1016/j.fertnstert.2009.12.001
The low implantation rate could be explained either by the in vitro aging of cultured oocytes before ICSI or by the asynchrony between the embryo development stage and the endometrial secretory pattern. To address this issue, we retrospectively reviewed a 5-year experience of rescue ICSI and analyzed the cycle outcome following transfer of fresh and frozen embryos. This retrospective study analyzed cases of unexplained failure of fertilization after cIVF observed in our infertility center between May 2004 and July 2009. Ovarian stimulations and cumulus–oocytes complexes retrieval were performed by standard procedures as previously described (11). Cumulus–oocytes complexes were routinely inseminated 3 to 5 hours after pickup with 10,000 motile selected spermatozoa in 30-mL appropriate embryo culture medium microdroplets under mineral oil and examined for evidence of fertilization 18 hours after insemination. Oocytes were considered normally fertilized when two pronuclei (2 PN) and two polar bodies were observed. The diploid fertilization rate was defined by the ratio of 2 PN and metaphase two oocytes. Metaphase two oocytes were considered as unfertilized when no pronucleus and only one polar body were observed into the ooplasm. In the case of complete cIVF fertilization failure, ICSI of unfertilized mature oocytes was performed as previously described (12) on day 1 with sperm from the original day 0 insemination. All included patients signed an informed consent form before rescue ICSI procedure. Oocytes were examined for evidence of fertilization 18 hours after ICSI and embryo quality was evaluated 48/72 hours after ICSI, according to previously described criteria (13). Good-quality embryos were transferred after enzymatic assisted hatching (AH) on day 3 after rescue ICSI. Good-quality supernumerary embryos were cryopreserved with a slow freezing protocol and transferred after AH in a subsequent thawing cycle. A clinical pregnancy was assessed by a positive fetal heartbeat on transvaginal ultrasound at 5 to 6 weeks following ET. Ongoing pregnancy was defined by an intrauterine pregnancy older than 12 weeks of amenorrhea. Data are presented as mean SD. Seventeen couples who experienced complete fertilization failure after cIVF provided written consent for a rescue ICSI procedure after receiving detailed information. Couple infertility included tubal factor (n ¼ 6), severe endometriosis (n ¼ 4),
Fertility and Sterility Vol. 94, No. 3, August 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
1157
ovulatory dysfunction (n ¼ 1), or idiopathic infertility (n ¼ 6). Women were 27 to 42 years old (35.5 3.6 years). Infertility duration was of 4.7 2.4 years. Basal blood FSH and antral follicle count determined at day 3 of a natural cycle was 7.5 2.1 IU/mL and 13.55 7.4, respectively. Controlled ovarian stimulation (COS) was performed using either a standard long GnRH agonist (n ¼ 8) or a GnRH antagonist (n ¼ 9) protocols. The total amount of rFSH administered was 2,219 1,069 IU and the duration of COS was 12.3 3.5 days. A mean of 7.47 3.53 unfertilized mature oocytes were microinjected. The 2 PN fertilization rate was 60.9%, whereas abnormal (R3 PN) fertilization rate was observed in 7.8% of injected oocytes. The percentage of good-quality embryo at the time of ET was of 70.6%. Fifteen fresh ETs of 2.35 1.17 embryos and 6 cryopreservations of 3.33 1.37 good-quality embryos were performed. After fresh ET, 1 ongoing pregnancy was obtained (pregnancy rate [PR] per ET ¼ 6.7%, per rescue ICSI cycle ¼ 5.9%). After embryo thawing (n ¼ 5 cycles), the survival rate was 85.7%, leading to five ETs of 2.40 0.55 embryos. Two ongoing pregnancies were obtained (PR per ET ¼ 40%, per thawing cycle ¼ 40%). One pregnancy led to the live birth after vaginal delivery of a healthy baby girl of 3,900 g weight at 39 weeks of gestation without complications. The other pregnancy is still ongoing after 20 weeks of gestation. This retrospective analysis of rescue ICSI shows that the PR is much higher following transfer of frozen–thawed good-quality embryos than after transfer of fresh embryos. The fertilization rate following ICSI of 1-day-old oocytes did not differ from other publications (5). However, the 3 PN fertilization rate after rescue ICSI was actually increased by 3.25-fold compared with that observed when oocytes are injected at day 0 (personnel data from 2 years consecutive ICSI results; 7.8% vs. 2.4%; P¼.0003). This result confirms previous reports (6–7, 10), and may be related to an abnormal ageing of oocytes in culture. In our study, a poor clinical outcome was observed following rescue ICSI procedure and
fresh ET. These data are in good agreement with previous studies reporting a large range of clinical PR (6.9% to 20.7%) (5–7) or even negative outcome (2, 10). In vitro aging of the oocyte could be responsible for this disappointing outcome after rescue ICSI (2). Indeed, a dramatic increase in cytogenetic abnormalities has been reported when the time interval between oocyte retrieval and fertilization is extended (9, 14). An alternative factor likely to contribute to the low PR following fresh ET get from rescue ICSI could be the asynchrony between embryo development and the endometrial secretory pattern. The endometrium is known to be receptive during a limited period of time, called the ‘‘implantation window’’ (15). Previous studies have shown that endometrial maturation according to Noyes’ criteria on the day of oocyte retrieval in IVF cycles is in advance by 2 to 4 days compared with natural cycles, regardless of controlled ovarian hyperstimulation protocols. In those studies, no ongoing clinical pregnancy was obtained if the endometrial advancement exceeds 3 days (16, 17). The procedure of day 1 rescue ICSI is likely to worsen this phenomenon by delaying the time of transfer. The promising ongoing PR observed in subsequent frozen–thawed cycles is in agreement with this assumption. Our results suggest that [1] the asynchrony between the embryo development and the endometrial receptivity could be the main contributing factor for low implantation rates observed after fresh ETs, and [2] that embryo cryopreservation with subsequent transfer in frozen–thawed cycles should be recommended to improve endometrial synchronization. In conclusion, day 1 rescue ICSI followed by embryo freezing could be a helpful tool to prevent from complete fertilization failure after cIVF and achieve live birth. Although prospective studies are needed to confirm these data, a systematic cryopreservation of all good-quality embryos obtained after rescue ICSI of unfertilized oocytes in cIVF could be an interesting option to better synchronize embryo development and endometrial receptivity.
REFERENCES 1. Mahutte NG, Arici A. Failed fertilization: is it predictable? Curr Opin Obstet Gynecol 2003;15:211–8. 2. Kuczynski W, Dhont M, Grygoruk C, Pietrewicz P, Redzko S, Szamatowicz M. Rescue ICSI of unfertilized oocytes after IVF. Hum Reprod 2002;17: 2423–7. 3. Lombardi E, Tiveron M, Inza R, Valcarcel A, Young E, Bisioli C. Live birth and normal 1-year follow-up of a baby born after transfer of cryopreserved embryos from rescue intracytoplasmic sperm injection of 1-day-old oocytes. Fertil Steril 2003;80:646–8. 4. Nagy ZP, Joris H, Liu J, Staessen C, Devroey P, Van Steirteghem AC. Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes. Hum Reprod 1993;8:2180–4. 5. Yuzpe AA, Liu Z, Fluker MR. Rescue intracytoplasmic sperm injection (ICSI)-salvaging in vitro fertilization (IVF) cycles after total or near-total fertilization failure. Fertil Steril 2000;73:1115–9. 6. Lundin K, Sjogren A, Hamberger L. Reinsemination of one-day-old oocytes by use of intracytoplasmic sperm injection. Fertil Steril 1996;66:118–21. 7. Morton PC, Yoder CS, Tucker MJ, Wright G, Brockman WD, Kort HI. Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes
1158
Sermondade et al.
8.
9.
10.
11.
12.
13.
after complete conventional fertilization failure. Fertil Steril 1997;68:488–91. Nagy Z, Staessen C, Liu J, Joris H, Devroey P, Van Steirteghem A. Prospective, auto-controlled study on reinsemination of failed-fertilized oocytes by intracytoplasmic sperm injection. Fertil Steril 1995;64:1130–5. Pehlivan T, Rubio C, Ruiz A, Navarro J, Remohi J, Pellicer A, et al. Embryonic chromosomal abnormalities obtained after rescue intracytoplasmic sperm injection of 1-day-old unfertilized oocytes. J Assist Reprod Genet 2004;21:55–7. Chen C, Kattera S. Rescue ICSI of oocytes that failed to extrude the second polar body 6 h post-insemination in conventional IVF. Hum Reprod 2003;18:2118–21. Huirne JA, Hugues JN, Pirard C, Fischl F, Sage JC, Pouly JL, et al. Cetrorelix in an oral contraceptivepretreated stimulation cycle compared with buserelin in IVF/ICSI patients treated with r-hFSH: a randomized, multicentre, phase IIIb study. Hum Reprod 2006;21:1408–15. Palermo G, Joris H, Devroey P, Van Steirteghem A. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17–8. Leniaud L, Poncelet C, Porcher R, Martin-Pont B, Cedrin-Durnerin I, Hugues J, et al. [Prospective
Rescue ICSI and embryo cryopreservation
14.
15.
16.
17.
evaluation of elective single-embryo transfer versus double-embryo transfer following in vitro fertilization: a two-year French hospital experience]. Gynecol Obstet Fertil 2008;36:159–65. DeUgarte CM, Li M, Jordan B, Hill D, DeCherney A and Surrey M. Rescue intracytoplasmic sperm injection and preimplantation genetic diagnosis in combination can result in pregnancy. Fertil Steril 2006;86: 200–2. Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 1999;340:1796–9. Kolibianakis E, Bourgain C, Albano C, Osmanagaoglu K, Smitz J, Van Steirteghem A, et al. Effect of ovarian stimulation with recombinant follicle-stimulating hormone, gonadotropin releasing hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil Steril 2002;78:1025–9. Ubaldi F, Bourgain C, Tournaye H, Smitz J, Van Steirteghem A, Devroey P. Endometrial evaluation by aspiration biopsy on the day of oocyte retrieval in the embryo transfer cycles in patients with serum progesterone rise during the follicular phase. Fertil Steril 1997;67:521–6.
Vol. 94, No. 3, August 2010