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Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer
FERTILITY AND STERILITY威 VOL. 77, NO. 1, JANUARY 2002 Copyright ©2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer Monozygotic twin pregnancy resulting after in vitro fertilization (IVF) treatment was reported for the first time by Yovich et al., (1). Although the natural rate of monozygotic twins (MZT) was reported to be 0.42% (2), the frequency after conventional IVF or intracytoplasmic sperm injection (ICSI) has been reported to vary between 1.2% and 8.9% (3–7). A significant increase in the incidence of MZT has been also reported after embryo transfer of day 5– 6 blastocysts compared with day 2–3 cleavage stage embryos (8). In this study, a total of 135 patients in 181 cycles treated by conventional IVF or ICSI were evaluated retrospectively between January 1999 and July 2000. Institutional Review Board approval was not obtained, since this is a retrospective and observational analysis. Indications for IVF were tubal disease and unexplained and multifactorial infertility, while male factor (azoospermia, cryptozoospermia and severe oligo-asthenoteratospermia) infertility was the main indication for ICSI. Patients were classified into two groups according to the type of the procedure. Group 1 included 48 patients treated by conventional IVF in 79 cycles in which 151 blastocysts were transferred, and Group 2 included 87 patients treated by ICSI in 102 cycles in which 310 blastocysts were transferred. No cases with assisted hatching were included in Group 1. The ovarian stimulation protocols used, patient monitoring, human chorionic gonadotropin (hCG) administration, and oocyte retrieval were described in details elsewhere (9). Address for correspondence (proofs and reprints): Basil C. Tarlatzis, M.D., Ph.D. Infertility & IVF Center, Geniki Kliniki 2, Gravias Street, Thessaloniki 546 45 Greece (FAX: ⫹30.31.821420 E-mail: [email protected]). 0015-0282/02/$22.00 PII S0015-0282(01)02958-2
196
The overall incidence of MZT was 3.3% per cycle. A statistically significant increase in the rates of multiple pregnancies (10.8% vs 2.6%) and MZT (5.9% vs 0) was observed in the ICSI group compared with the conventional IVF group (P ⫽ 0.039 and P ⫽ 0.033, respectively). On the other hand, there were no significant differences between the two groups in the
incidence of high-order multiple gestations, selective reductions, miscarriages, or ectopic pregnancies (Table 1). In the conventional IVF group, 2 patients had quadruplet pregnancies, and both of them underwent selective reduction. There were no cases of monozygosity in this group. In the ICSI group, there were 1 quadruplet, 5 triplet (including one set of monozygotic twins) and 5 twin pregnancies. Selective reduction was performed in 5 cases. Interestingly, all cases of monozygotic twins were monochorionic-diamniotic, as documented by vaginal ultrasound, which was performed 6 –7 weeks after the blastocyst embryo transfer. Although the mechanism by which monozygotic twins are formed is not completely known, many investigators have postulated that the splitting of the inner cell mass at an early stage of development may cause the duplication of the embryo (4, 6, 7). Splitting of the zygote may occur at any time during the first 2 weeks after fertilization, resulting in the various forms of monozygotic twins (7). In about one-third of MZT, the zygotic division occurs within 72 hours of fertilization and the placenta will be dichorionic-diamniotic, similar to that of dizygotic twins. In approximately twothirds, the zygotic division may occur 4 to 8 days after fertilization and the placenta will be monochorionic-diamniotic. In approximately 5% of cases, the division occurs 8 –13 days after fertilization resulting in monochorionicmonoamniotic twins. The division rarely occurs after 13 days; when it does occur, the result will be conjoined twins. Factors that appear to predispose to this splitting include alterations in the thickness of zona pellucida, time of implantation, and the assisted reproduction techniques (ART). The micromanipulation of zona pellucida in assisted reproduction techniques (including subzonal insemination, assisted hatching, and
TABLE 1 Clinical data and outcome of 79 conventional IVF cycles and 102 ICSI cycles (mean ⫾ SD).
No. of patients No. of cycles No. of cancelled cycles Age of patients (y) No. of oocytes retrieved No. of blastocysts transferred Fertilization rate (%) Implantation rate (%) Pregnancy rate/cycle (%) Multiple pregnancy rate/cycle (%) High-order multiple pregnancy rate/cycle (%) Monozygotic twin rate/cycle (%) Abortion rate/cycle (%) Reduction rate/cycle (%) Ectopic pregnancy rate/cycle (%)
Conventional IVF
ICSI
P
48 79 3 31.9 ⫾ 2.9 9.78 ⫾ 4.1 2.78 ⫾ 1.48 71.5 13.9 15/76 (19.7) 2/76 (2.6 ) 2/76 (2.6 ) 0 1/76 (1.3 ) 2/76 (2.6 ) 0
87 102 0 31.8 ⫾ 4.1 13.4 ⫾ 5.07 3.19 ⫾ 1.6 70 16.5 33/102 (32.5) 11/102 (10.8) 6/102 (5.9 ) 6/102 (5.9 ) 6/102 (5.9 ) 5/102 (4.9 ) 2/102 (1.9 )
NS NS NS NS NS NS .039 NS .033 NS NS NS
Note: NS ⫽ not significant. Tarlatzis. Blastocyst and monozygote twinning rate. Fertil Steril 2002.
ICSI) has been reported to increase the frequency of MZT (3–5, 7). An artificial opening introduced in the zona pellucida caused by the use of these procedures may alter the process of hatching, while the expansion during expulsion of the blastocyst through the artificial opening may cause constriction and subsequent bisection of the trophoblast and inner cell mass, which could result in twinning (4, 10). A single artificial opening in the zona pellucida may lead to a figure-eight configuration at the time of hatching leading to MZT, while multiple holes that are caused sometimes by repeated manipulation of the zona will promote multiple herniation, resulting in unexpected splitting of the inner cell mass, since monozygotic triplets have also been reported (5, 11). Our findings are consistent with those presented by Abusheika and colleagues (7); they reported an incidence of 8.9% of MZT subsequent to ICSI treatment compared with 0.9% after conventional IVF treatment. The effect of zona pellucida manipulation on MZT was further confirmed by another study that showed significant increase in the rate of MZT after mechanical assisted hatching (1.2%) as compared with nonhatching (5). These findings are probably due to the artificial opening created by mechanical trauma (assisted hatching or ICSI) rather than by natural autolysis of the zona pellucida which is responsible for splitting of the inner cell mass, since there were no cases of MZT in the nonmanipulated zona pellucidae. It is of interest that all MZT reported in our series as well as in other studies (5, 7) were monochorionic-diamniotic. This is probably related to the timing of embryo division. As mentioned above, the monochorionic-diamniotic twins are formed after splitting of the inner cell mass, that occurs 4 to FERTILITY & STERILITY威
8 days after fertilization, and, therefore, the embryonic division would take place near the time of implantation. The monozygotic twin pregnancies are associated with an increased morbidity and mortality for the embryos and the neonates (12). In the present study, one patient delivered by cesarean section, one delivered prematurely, two patients miscarried in the second trimester, and two pregnancies are ongoing. Our findings indicate that the incidence of MZT is increased significantly among patients undergoing embryo transfer in the blastocyst stage after the application of ICSI. Splitting of the inner cell mass caused by the artificial opening induced by the procedure may be the responsible mechanism. Monozygotic twins formed in this manner tend to be monochorionic-diamniotic. Basil C. Tarlatzis, M.D., Ph.D. Hussein S. Qublan, M.D. Thomal Sanopoulou, M.S. Leonides Zepiridis, M.D. Gregoris Grimbizis, M.D. John Bontis, M.D., Ph.D. Infertility & IVF Center, Geniki Kliniki and Unit of Human Reproduction, 1st Department of Obstetrics and Gynaecology, Aristotle University of Thessaloniki, Thessaloniki, Greece
References 1. Yovich JL, Stanger JD, Gravaug A, Barter RA, Lunay G, Dawkins RL, et al. Monozygotic twins from in vitro fertilization. Fertil Steril 1984; 41:833–7. 2. Bulmer MG. The biology of twinning in man. Oxford: Clarendon press, 1970. 3. Edwards RG, Mettler L, Walter DE. Identical twins and in vitro fertilization. J In Vitro Fertil Embryo Transfer 1986;3:114 –7. 4. Alikani M, Noyes N, Cohen J, Rosenwaks Z. Monozygotic twinning in human is associated with zona pellucida architecture. Hum Reprod 1994;9:1318 –21. 5. Hershlag A, Paine T, Cooper GW, Scholl GM, Rawlinson K, Kvapil G. Monozygotic twinning associated with mechanical assisted hatching. Fertil. Steril 1999;71:144 – 6.
197
6. Sills E, Rosenwaks Z, Moomjy M, Zaninovic N, Veeck McGee LL, Palermo GD. Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF. Hum Reprod 2000;15:890 –5. 7. Abusheika N, Salha O, Sharma V, Brinsden P. Monozygotic twinning and IVF/ICSI treatment: a report of 11 cases and review of literature. Hum Reprod Update 2000;6:396 –3. 8. Rijnders PM, Van Os HC, Jansen CAM. Increased incidence of monozygotic twinning following the transfer of blastocysts in human IVF/ICSI. Fertil Steril 1998;70:S 15– 6. 9. Tarlatzis BC, Grimbizis G, Pournaropoulos F, Bontis J, Lagos S, Pados G et al. Evaluation of two gonadotropin-releasing hormone
198
Tarlatzis
Correspondence
(GnRH) analogues (Leuprolide and Buserelin) in short and long protocols for assisted reproduction techniques. J Assist Reprod Genet 1994;11:85–9. 10. Cohen J, Malter H, Wright G, Kort H, Massey J, et al. Partial zona dissection of human oocyte when failure of zona pellucida penetration is anticipated. Hum Reprod 1989;4:435–2. 11. Belaish-Allart J, Elaoufir A, Mayenga JM, Segard L, Bernard JP, Plachot M. Monozygotic triplet pregnancy after transfer of frozen embryos. Contraception Fertilite Sexualite 1995;23:435–2. 12. Cohen J. How to avoid multiple pregnancies in assisted reproduction. Hum Reprod 1998;13 (Suppl. 3):197-4.
Vol. 77, No. 1, January 2002
Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer Monozygotic twin pregnancy resulting after in vitro fertilization (IVF) treatment was reported for the first time by Yovich et al., (1). Although the natural rate of monozygotic twins (MZT) was reported to be 0.42% (2), the frequency after conventional IVF or intracytoplasmic sperm injection (ICSI) has been reported to vary between 1.2% and 8.9% (3–7). A significant increase in the incidence of MZT has been also reported after embryo transfer of day 5– 6 blastocysts compared with day 2–3 cleavage stage embryos (8). In this study, a total of 135 patients in 181 cycles treated by conventional IVF or ICSI were evaluated retrospectively between January 1999 and July 2000. Institutional Review Board approval was not obtained, since this is a retrospective and observational analysis. Indications for IVF were tubal disease and unexplained and multifactorial infertility, while male factor (azoospermia, cryptozoospermia and severe oligo-asthenoteratospermia) infertility was the main indication for ICSI. Patients were classified into two groups according to the type of the procedure. Group 1 included 48 patients treated by conventional IVF in 79 cycles in which 151 blastocysts were transferred, and Group 2 included 87 patients treated by ICSI in 102 cycles in which 310 blastocysts were transferred. No cases with assisted hatching were included in Group 1. The ovarian stimulation protocols used, patient monitoring, human chorionic gonadotropin (hCG) administration, and oocyte retrieval were described in details elsewhere (9). Address for correspondence (proofs and reprints): Basil C. Tarlatzis, M.D., Ph.D. Infertility & IVF Center, Geniki Kliniki 2, Gravias Street, Thessaloniki 546 45 Greece (FAX: ⫹30.31.821420 E-mail: [email protected]). 0015-0282/02/$22.00 PII S0015-0282(01)02958-2
196
The overall incidence of MZT was 3.3% per cycle. A statistically significant increase in the rates of multiple pregnancies (10.8% vs 2.6%) and MZT (5.9% vs 0) was observed in the ICSI group compared with the conventional IVF group (P ⫽ 0.039 and P ⫽ 0.033, respectively). On the other hand, there were no significant differences between the two groups in the
incidence of high-order multiple gestations, selective reductions, miscarriages, or ectopic pregnancies (Table 1). In the conventional IVF group, 2 patients had quadruplet pregnancies, and both of them underwent selective reduction. There were no cases of monozygosity in this group. In the ICSI group, there were 1 quadruplet, 5 triplet (including one set of monozygotic twins) and 5 twin pregnancies. Selective reduction was performed in 5 cases. Interestingly, all cases of monozygotic twins were monochorionic-diamniotic, as documented by vaginal ultrasound, which was performed 6 –7 weeks after the blastocyst embryo transfer. Although the mechanism by which monozygotic twins are formed is not completely known, many investigators have postulated that the splitting of the inner cell mass at an early stage of development may cause the duplication of the embryo (4, 6, 7). Splitting of the zygote may occur at any time during the first 2 weeks after fertilization, resulting in the various forms of monozygotic twins (7). In about one-third of MZT, the zygotic division occurs within 72 hours of fertilization and the placenta will be dichorionic-diamniotic, similar to that of dizygotic twins. In approximately twothirds, the zygotic division may occur 4 to 8 days after fertilization and the placenta will be monochorionic-diamniotic. In approximately 5% of cases, the division occurs 8 –13 days after fertilization resulting in monochorionicmonoamniotic twins. The division rarely occurs after 13 days; when it does occur, the result will be conjoined twins. Factors that appear to predispose to this splitting include alterations in the thickness of zona pellucida, time of implantation, and the assisted reproduction techniques (ART). The micromanipulation of zona pellucida in assisted reproduction techniques (including subzonal insemination, assisted hatching, and
TABLE 1 Clinical data and outcome of 79 conventional IVF cycles and 102 ICSI cycles (mean ⫾ SD).
No. of patients No. of cycles No. of cancelled cycles Age of patients (y) No. of oocytes retrieved No. of blastocysts transferred Fertilization rate (%) Implantation rate (%) Pregnancy rate/cycle (%) Multiple pregnancy rate/cycle (%) High-order multiple pregnancy rate/cycle (%) Monozygotic twin rate/cycle (%) Abortion rate/cycle (%) Reduction rate/cycle (%) Ectopic pregnancy rate/cycle (%)
Conventional IVF
ICSI
P
48 79 3 31.9 ⫾ 2.9 9.78 ⫾ 4.1 2.78 ⫾ 1.48 71.5 13.9 15/76 (19.7) 2/76 (2.6 ) 2/76 (2.6 ) 0 1/76 (1.3 ) 2/76 (2.6 ) 0
87 102 0 31.8 ⫾ 4.1 13.4 ⫾ 5.07 3.19 ⫾ 1.6 70 16.5 33/102 (32.5) 11/102 (10.8) 6/102 (5.9 ) 6/102 (5.9 ) 6/102 (5.9 ) 5/102 (4.9 ) 2/102 (1.9 )
NS NS NS NS NS NS .039 NS .033 NS NS NS
Note: NS ⫽ not significant. Tarlatzis. Blastocyst and monozygote twinning rate. Fertil Steril 2002.
ICSI) has been reported to increase the frequency of MZT (3–5, 7). An artificial opening introduced in the zona pellucida caused by the use of these procedures may alter the process of hatching, while the expansion during expulsion of the blastocyst through the artificial opening may cause constriction and subsequent bisection of the trophoblast and inner cell mass, which could result in twinning (4, 10). A single artificial opening in the zona pellucida may lead to a figure-eight configuration at the time of hatching leading to MZT, while multiple holes that are caused sometimes by repeated manipulation of the zona will promote multiple herniation, resulting in unexpected splitting of the inner cell mass, since monozygotic triplets have also been reported (5, 11). Our findings are consistent with those presented by Abusheika and colleagues (7); they reported an incidence of 8.9% of MZT subsequent to ICSI treatment compared with 0.9% after conventional IVF treatment. The effect of zona pellucida manipulation on MZT was further confirmed by another study that showed significant increase in the rate of MZT after mechanical assisted hatching (1.2%) as compared with nonhatching (5). These findings are probably due to the artificial opening created by mechanical trauma (assisted hatching or ICSI) rather than by natural autolysis of the zona pellucida which is responsible for splitting of the inner cell mass, since there were no cases of MZT in the nonmanipulated zona pellucidae. It is of interest that all MZT reported in our series as well as in other studies (5, 7) were monochorionic-diamniotic. This is probably related to the timing of embryo division. As mentioned above, the monochorionic-diamniotic twins are formed after splitting of the inner cell mass, that occurs 4 to FERTILITY & STERILITY威
8 days after fertilization, and, therefore, the embryonic division would take place near the time of implantation. The monozygotic twin pregnancies are associated with an increased morbidity and mortality for the embryos and the neonates (12). In the present study, one patient delivered by cesarean section, one delivered prematurely, two patients miscarried in the second trimester, and two pregnancies are ongoing. Our findings indicate that the incidence of MZT is increased significantly among patients undergoing embryo transfer in the blastocyst stage after the application of ICSI. Splitting of the inner cell mass caused by the artificial opening induced by the procedure may be the responsible mechanism. Monozygotic twins formed in this manner tend to be monochorionic-diamniotic. Basil C. Tarlatzis, M.D., Ph.D. Hussein S. Qublan, M.D. Thomal Sanopoulou, M.S. Leonides Zepiridis, M.D. Gregoris Grimbizis, M.D. John Bontis, M.D., Ph.D. Infertility & IVF Center, Geniki Kliniki and Unit of Human Reproduction, 1st Department of Obstetrics and Gynaecology, Aristotle University of Thessaloniki, Thessaloniki, Greece
References 1. Yovich JL, Stanger JD, Gravaug A, Barter RA, Lunay G, Dawkins RL, et al. Monozygotic twins from in vitro fertilization. Fertil Steril 1984; 41:833–7. 2. Bulmer MG. The biology of twinning in man. Oxford: Clarendon press, 1970. 3. Edwards RG, Mettler L, Walter DE. Identical twins and in vitro fertilization. J In Vitro Fertil Embryo Transfer 1986;3:114 –7. 4. Alikani M, Noyes N, Cohen J, Rosenwaks Z. Monozygotic twinning in human is associated with zona pellucida architecture. Hum Reprod 1994;9:1318 –21. 5. Hershlag A, Paine T, Cooper GW, Scholl GM, Rawlinson K, Kvapil G. Monozygotic twinning associated with mechanical assisted hatching. Fertil. Steril 1999;71:144 – 6.
197
6. Sills E, Rosenwaks Z, Moomjy M, Zaninovic N, Veeck McGee LL, Palermo GD. Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF. Hum Reprod 2000;15:890 –5. 7. Abusheika N, Salha O, Sharma V, Brinsden P. Monozygotic twinning and IVF/ICSI treatment: a report of 11 cases and review of literature. Hum Reprod Update 2000;6:396 –3. 8. Rijnders PM, Van Os HC, Jansen CAM. Increased incidence of monozygotic twinning following the transfer of blastocysts in human IVF/ICSI. Fertil Steril 1998;70:S 15– 6. 9. Tarlatzis BC, Grimbizis G, Pournaropoulos F, Bontis J, Lagos S, Pados G et al. Evaluation of two gonadotropin-releasing hormone
198
Tarlatzis
Correspondence
(GnRH) analogues (Leuprolide and Buserelin) in short and long protocols for assisted reproduction techniques. J Assist Reprod Genet 1994;11:85–9. 10. Cohen J, Malter H, Wright G, Kort H, Massey J, et al. Partial zona dissection of human oocyte when failure of zona pellucida penetration is anticipated. Hum Reprod 1989;4:435–2. 11. Belaish-Allart J, Elaoufir A, Mayenga JM, Segard L, Bernard JP, Plachot M. Monozygotic triplet pregnancy after transfer of frozen embryos. Contraception Fertilite Sexualite 1995;23:435–2. 12. Cohen J. How to avoid multiple pregnancies in assisted reproduction. Hum Reprod 1998;13 (Suppl. 3):197-4.
Vol. 77, No. 1, January 2002