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Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer
FERTILITY AND STERILITY威 VOL. 79, NO. 3, MARCH 2003 Copyright ©2003 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
IN VITRO FERTILIZATION
Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer Amin A. Milki, M.D., Sunny H. Jun, M.D., Mary D. Hinckley, M.D., Barry Behr, Ph.D., Linda C. Giudice, M.D., and Lynn M. Westphal, M.D. Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California
Objective: To evaluate the incidence of monozygotic twinning (MZT) in pregnancies conceived after blastocyst transfer compared to cleavage-stage transfer. Design: Retrospective study. Setting: University IVF program. Patient(s): All IVF patients with viable pregnancies conceived during a 4-year period. Intervention(s): Blastocyst transfer or day 3 ET. Main Outcome Measure(s): Incidence of MZT assessed by transvaginal ultrasound. Result(s): There were 11 incidences of MZT in 197 viable pregnancies (5.6%) with blastocyst transfer compared to 7 of 357 viable pregnancies (2%) with day 3 ET. In 10 of 18 pregnancies, MZT was observed in the setting of a higher order multiple gestation (6 of 11 for blastocyst transfer and 4 of 7 for day 3 ET). In the day 3 ET group, assisted hatching or intracytoplasmic sperm injection (ICSI) did not increase MZT (4 of 213, 1.9%) compared to cycles without zona breaching (3 of 144, 2.1%). Similarly, in the blastocyst-transfer group, ICSI did not increase the incidence of MZT (4 of 74, 5.5% for ICSI and 7 of 123, 5.7% for non-ICSI IVF). Conclusion(s): Compared to day 3 ET, blastocyst transfer appears to significantly increase the incidence of gestations with MZT. This information should be taken into account when counseling patients about the pros and cons of extended culture. (Fertil Steril威 2003;79:503– 6. ©2003 by American Society for Reproductive Medicine.) Key Words: Blastocyst transfer, monozygotic twinning, cleavage stage transfer, multiple gestations, IVF
Received July 5, 2002; revised and accepted September 9, 2002. Presented at the 58th Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, October 14 –17, 2002. Reprint requests: Amin A. Milki, M.D., Department of Gynecology and Obstetrics, Stanford University School of Medicine, 300 Pasteur Drive, HH333, Stanford, California 94305 (FAX: 650498-7294; E-mail: [email protected]). 0015-0282/03/$30.00 doi:10.1016/S0015-0282(02) 04754-4
Monozygotic twinning (MZT) occurs in 0.42% of all births (1). An increased incidence of MZT has been reported in pregnancies conceived after ovulation induction (2) and IVF (3–7), especially when the zona pellucida is breached (4, 5, 8 –10). Although blastocyst transfer can be used to prevent higher order multiple gestations by limiting the transfer to two embryos (11–15), several studies have raised the possibility that MZT is more common with blastocyst transfer (8, 16 –18). However, only two published reports in the literature have specifically assessed the incidence of MZT with blastocyst transfer compared to day 3 ET (17, 18). One of these studies involved only nine pregnancies after blastocyst transfer (17). The other study (18), which looked exclusively at intracytoplasmic sperm injection (ICSI) cycles, compared pregnancies resulting
from blastocyst transfer with those conceived with day 3 ET during the study period and the previous 3 years. The purpose of our study is to compare the incidence of MZT in a large series of day 3 and day 5 ET performed in IVF and ICSI cycles completed during the same time period.
MATERIALS AND METHODS We retrospectively analyzed all viable pregnancies conceived in our IVF program since January 1998, when blastocyst transfer was introduced in our center. The incidence of MZT was examined in pregnancies resulting from blastocyst transfer compared to day 3 ET during this time period. The controlled ovarian hyperstimulation protocol consisted of pretreatment with oral 503
contraceptive (OC) pills with overlapping GnRH agonist down-regulation followed by FSH/hMG and hCG or a microdose flare protocol. Oocytes were inseminated conventionally or by ICSI 3– 4 hours after retrieval. For ICSI, oocytes were denuded using a hyaluronidase solution combined with mechanical stripping. Oocytes were then rinsed and those at the metaphase II stage were injected in phosphate-buffered saline (PBS) with 10% supplement serum substitute (SSS) (Irvine Scientific, Santa Ana, CA). Embryos were cultured in groups under mineral oil in 150-L droplets of P1 medium (Irvine Scientific) with 10% SSS at 37°C in a 5% O2, 5% CO2, and 90% N2 environment for 72 hours. In selected cases, assisted hatching was performed before day 3 ET. Embryos for hatching were placed in PBS with 10% SSS. A small hole (⬍20 m) was made using acidified Tyrode’s solution (pH 2.3) in an area of the zona pellucida that was between blastomeres. Embryos were then rinsed and returned to culture until ET. For the blastocyst transfer group, the embryos were moved on day 3 into blastocyst medium (Irvine Scientific) with 10% SSS and cultured for an additional 48 hours before transfer. All transfers were performed using a Tefcat catheter (Cook Ob/Gyn, Spencer, IN). A viable pregnancy was defined as the presence of cardiac activity confirmed by transvaginal ultrasound at 7 weeks’ gestation. The number of fetuses and gestational sacs was assessed. Monozygotic twinning was diagnosed when more than one fetus with cardiac activity was seen in the same gestational sac. There was no incidence of more gestational sacs than number of embryos transferred. Also, the presence or absence of a dividing amnionic membrane was noted. The findings were reconfirmed with both a repeat ultrasound examination 2 weeks later and follow-up information for the rest of the pregnancy and delivery. Statistical analysis of results was performed using 2 testing. Significance was set at P⬍.05. Institutional review board approval was obtained for review of patient charts.
RESULTS There were 11 incidences of MZT in 197 viable pregnancies (5.6%) with blastocyst transfer compared to 7 of 357 viable pregnancies (2%) with day 3 ET (P⬍.03). Of 127 pregnancies with multiple gestational sacs seen in the entire study population, there were 10 incidences of MZT (6 from blastocyst transfer and 4 from day 3 ET) compared to 8 of 427 pregnancies with a single gestational sac (7.9% vs. 1.9%, P⬍.003). There was no significant difference in the overall multiple gestation rate between the blastocyst transfer group (43 of 197, 22%) and the day 3 ET group (84 of 357, 24%). In the day 3 ET group, the incidence of MZT was not increased by assisted hatching ⫾ ICSI (3 of 155, 1.9%) or ICSI alone (1 of 58, 1.7%) compared to cycles without zona breaching (3 of 144, 2.1%). Similarly, in the blastocyst transfer group, where assisted hatching was not performed, 504
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ICSI did not increase the incidence of MZT (4 of 74, 5.5% for ICSI and 7 of 123, 5.7% for non-ICSI IVF).
DISCUSSION The exact mechanism leading to MZT remains unclear. Factors such as maternal age, family history, race, and parity, which have been associated with dizygotic twinning, do not seem to affect the overall frequency of MZT, which has been reported at 0.42% of all births in the general population (1). It is likely that this incidence would be higher if early first trimester pregnancies were examined. Of the 11 MZ twins reported in our blastocyst transfer pregnancies, 5 spontaneously resolved. Still, the remaining incidence of 3% (6 of 197) represents a sevenfold increase over the natural occurrence of MZT. Ideally, to account for dichorionic MZ twins, the incidence of MZT with IVF should be assessed by looking at transfers of a single embryo leading to a multiple pregnancy. Therefore, our method of identifying MZ twins may underestimate the actual occurrence of this problem by looking only at monochorionic MZ twins. However, these pregnancies are of more clinical relevance due to their known increased perinatal morbidity and mortality. In the context of infertility treatment, Edwards et al. (3) observed a higher than expected frequency of MZ splitting after IVF (1.3%). Derom et al. (2) found a 1.2% frequency of MZ splitting in patients exposed to ovulation induction, which was significantly higher than the expected frequency with spontaneous ovulation. They postulated that in IVF cycles, it is the ovulation induction itself, rather than in vitro conditions, which predisposes to zygotic division or to enhanced survival of MZ twins. Since these early studies, several investigators have reported an increased incidence of MZT in IVF pregnancies (4 –7, 9, 19 –22). Most of these reports have shown that MZT occurred more frequently when the zona pellucida was manipulated to perform assisted hatching, subzonal insemination (SUZI), or ICSI (4, 5, 7–9, 20, 23). These zona breaching techniques result in artificial openings that may interfere with the natural process of hatching. Depending on the actual size and number of these artificial gaps, the embryo may possibly herniate following the path of least resistance and result in a division of the inner cell mass, leading to the formation of identical twins. However, in a recent study, Blickstein et al. (21) showed no increase in MZT with ICSI. Similarly, Sills et al. (6) and Schachter et al. (22) reported no difference in the incidence of MZT in ICSI and assisted hatching cycles compared to IVF without zona breaching. Adding further support to the findings from the latter three studies, our own data did not show a difference in the frequency of MZT in pregnancies derived from ICSI or assisted hatching compared to conventional IVF. Further Vol. 79, No. 3, March 2003
studies of larger samples are still needed to shed light on the association of MZT with zona manipulation. Our study shows that MZT occurs more frequently in pregnancies conceived after blastocyst transfer compared to day 3 ET with an incidence of 5.6% and 2%, respectively. These findings add pertinent information to the literature comparing blastocyst with cleavage-stage transfer in an area that has not been adequately addressed. Initial data presented in an abstract by Rijnders et al. (24) suggested that blastocyst transfer was associated with a higher incidence of MZT compared to cleavage-stage transfer. However, the literature contains only one large published study by da Costa et al. (18), which examined the frequency of MZT in 129 day 5 ET compared to day 3 ET controls, all in ICSI cycles. The data from our study confirm that the increased MZT seen with blastocyst transfer compared to day 3 ET in ICSI cycles, as reported by da Costa et al., is also found in the general IVF population irrespective of whether ICSI was performed. Interestingly, we have found a 7.9% incidence of monozygotic splitting in pregnancies where two or more gestational sacs were present compared to 1.9% in pregnancies with a single gestational sac. This higher rate of MZT in the setting of multiple gestations confirms previous reports. Derom et al. (2) observed that the frequency of splitting was significantly higher in the triplets than in the twins conceived after ovulation induction. Similarly, Wenstrom et al. (19), who reported a 3.2% incidence of MZT in assisted reproduction pregnancies, showed a 9.8% monochorionicity rate in the multiple gestation patients. The overall incidence of multiple gestations in our study population was similar for both day 3 and day 5 ET and is unlikely to be a source of bias accounting for the higher rate of MZT encountered with blastocyst transfer. Herniation of the blastocyst through a less flexible zona, hardened by prolonged in vitro culture, has been proposed as a possible mechanism leading to MZT (4). However, Frankfurter et al. (25) showed a 1.2% MZT rate with zona free (pronase digested) blastocyst transfer, which was not significantly different from the rate seen with zona intact blastocyst transfer in their patients, suggesting that other etiologies must be present. Fertilization in vitro has been associated with a duplication of the inner cell mass (ICM). Chida (26) found that 3.1% of mouse blastocysts fertilized in vitro have a double ICM compared to 0.6% of in vivo fertilized blastocysts. Meintjes et al. (27) reported triplets after the transfer of two blastocysts with one showing two distinct ICMs. The depressed calcium level in a free blastocyst before implantation compared to that found in the endometrium in animal studies has been proposed by Steinman (28) as a mechanism for increased MZT by weakening ICM intercellular bonding and predisposing the embryo to division. This researcher suggests that in vitro incubation for 5 days rather FERTILITY & STERILITY威
than 3 days increases MZT by prolonging the exposure to lower calcium concentrations. However, it would have to be shown that embryos transferred on day 5 implant later than those transferred on day 3 for this theory to be valid. Recently, Menezo and Sakkas (29) proposed a possible explanation for the phenomenon of MZT in blastocyst transfer. They mention that they had no incidence of MZT in more than 800 deliveries when co-culture was used to obtain blastocysts. They believe that media used to grow blastocysts without co-culture may lead to an overstimulation of apoptosis through free radical formation induced by excessive glucose levels. Linear polarization of apoptotic cells in the ICM could lead to splitting during or before the hatching process. They suggest that co-culture using feeder cells may provide free radical scavengers more efficiently than current blastocyst culture media, which could account for the absence of MZT in their patients. In our study, blastocyst medium (Irvine Scientific), which contains 6 mM of glucose, was used. Other reports in the literature suggest that MZT does occur with different blastocyst media with lower glucose concentrations such as S2 or G2.2 (IVF Science, Gothenburg, Sweden). Behr et al. (16) in a multicenter study reported several occurrences of MZT with the use of S2 medium. Da Costa et al. (18) also used S2 for extended culture in their study showing increased MZT with blastocyst transfer. Schoolcraft and Gardner (30) reported two triplet pregnancies resulting from MZT in 89 ongoing pregnancies with blastocyst transfer using G2.2 medium in oocyte donation cycles, whereas Wilson et al. (31), using S2 or G2.2, found that 11 of 12 triplet births from day 5 ET were due to MZT, representing 3.7% of ongoing pregnancies. The true incidence of MZ splitting is very likely to be higher in the latter two studies, as MZT leading to twin gestations was not addressed. On the basis of some recent studies, an alternative explanation, which may account for the absence of MZT when co-culture is used to grow blastocysts, is the production of cytokines or growth factors by mitotically active cells rather than the presence of free radical scavengers. Free radical scavengers like glutathione or taurine are added to many culture media. In particular, high doses of glutathione are present in the blastocyst medium used in our study. Wang et al. (32) demonstrated that in the presence of the growth factor GM-CSF, mouse blastocysts had significantly less apoptotic cells compared to blastocysts grown in standard culture conditions. Behr et al. (33) also demonstrated that the presence of GM-CSF in the culture medium up-regulated the expression of Connexin-37, an integral tight junction protein. Therefore, it is possible that culture media devoid of growth factors or cytokines may impose metabolic stress on embryos that is exacerbated by extended culture. This may translate into higher rates of MZT due in part to increased apoptosis and less cell– cell adhesion. However, caution should be exercised in extrapolating the results from these 505
mouse studies. The data need to be confirmed in human embryos. In conclusion, our study shows that MZT is more common with blastocyst transfer compared to cleavage-stage transfer. Although blastocyst transfer allows the selection of the most viable embryos and accordingly, offers the potential to limit the number of embryos transferred, the reported increase in MZT has to be weighed against the ability of blastocyst transfer to reduce fraternal high order multiple births. The findings from our study should be considered when counseling patients about risks and benefits of extended culture. References 1. Bulmer MG. The biology of twinning in man. Oxford: Clarendon Press, 1970. 2. Derom C, Vlietinck R, Derom R, Van den Berghe H, Thiery M. Increased monozygotic twinning rate after ovulation induction. Lancet 1987;1:1236 –8. 3. Edwards RG, Mettler LE, Walters 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 the human is associated with the 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. 6. Sills ES, Moomjy M, Zaninovic N, Veeck LL, McGee M, Palermo GD, et al. Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF. Hum Reprod 2000;15:890 –5. 7. Abusheikha N, Salha O, Sharma V, Brinsden P. Monozygotic twinning and IVF/ICSI treatment: a report of 11 cases and review of the literature. Hum Reprod Update 2000;6:396 –403. 8. Tarlatzis BC, Qublan HS, Sanopoulou T, Zepiridis L, Grimbizis G, Bontis J. Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer. Fertil Steril 2002;72:196 –8. 9. Saito H, Tsutsumi O, Noda Y, Ibuki Y, Hiroi M. Do assisted reproductive technologies have effects on the demography of monozygotic twinning? Fertil Steril 2000;74:178 –9. 10. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer P, 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. 11. Gardner DK, Schoolcraft WB, Wagley L, Schenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998;13:3434 –40. 12. Milki AA, Fisch JD, Behr B. Two blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared to three blastocyst transfer. Fertil Steril 1999;72:225–8. 13. Karaki RZ, Samarraie SS, Younis NA, Lahloub TM, Ibrahim MH. Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome. Fertil Steril 2002;77:114 –8.
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14. Racowsky C, Jackson KV, Cekleniak NA, Fox JH, Hornstein MD, Ginsburg ES. The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. Fertil Steril 2000;73:558 –64. 15. Toledo AA, Wright G, Jones AE, Smith SS, Johnson-Ward J, Brockman WW, et al. Blastocyst transfer: a useful tool for reduction of high-order multiple gestations in a human assisted reproduction program. Am J Obstet Gynecol 2000;183:3777–9. 16. Behr B, Fisch JD, Racowsky C, Miller K, Pool TB, Milki AA. Blastocyst-ET and monozygotic twinning. J Assist Reprod Genet 2000;17: 349 –51. 17. Sheiner E, Har-Vardi I, Potashnik G. The potential association between blastocyst transfer and monozygotic twinning. Fertil Steril 2001;75: 217–8. 18. Da Costa AL, Abdelmassih S, de Oliveira FG, Abdelmassih V, Abdelmassih R, Nagy ZP, et al. Monozygotic twins and transfer at the blastocyst stage after ICSI. Hum Reprod 2001;16:333–6. 19. Wenstrom KD, Syrop CH, Hammit DG, VanVoorhis BJ. Increased risk of monochorionic twinning associated with assisted reproduction. Fertil Steril 1993;60:510 –4. 20. Slotnick RN, Ortega JE. Monoamniotic twinning and zona manipulation: a survey of US IVF centers correlating zona manipulation procedures and high-risk twinning frequency. J Assist Reprod Genet 1996; 13:381–5. 21. Blickstein I, Verhoeven HC, Keith LG. Zygotic splitting after assisted reproduction. N Engl J Med 1999;340:738 –9. 22. Schachter M, Raziel A, Friedler S, Strassburger D, Bern O, Ron-El R. Monozygotic twinning after assisted reproductive techniques: a phenomenon independent of micromanipulation. Hum Reprod 2001;16: 1264 –9. 23. Schieve LA, Meikle SF, Peterson HB, Jeng G, Burnett NM, Wilcox LS. Does assisted hatching pose a risk for monozygotic twinning in pregnancies conceived through in vitro fertilization? Fertil Steril 2000;74: 288 –94. 24. Rijnders PM, van Os HC, Jansen CA. Increased incidence of monozygotic twinning following the transfer of blastocysts in human IVF/ICSI. Fertil Steril 1998;70:15S–6S. 25. Frankfurter D, Hackett R, Meng L, Keefe DL. Complete removal of the zona pellucida by pronase digestion prior to blastocyst embryo transfer does not eliminate monozygotic pregnancies following IVF. Fertil Steril 2001;76:144S. 26. Chida S. Monozygous double inner cell masses in mouse blastocysts following fertilization in vitro and in vivo. J In Vitro Fert Embryo Transf 1990;7:177–9. 27. Meintjes M, Guerami AR, Rodriguez JA, Crider-Pirkle SS, Madden JD. Prospective identification of an in vitro-assisted monozygotic pregnancy based on a double-inner-cell-mass blastocyst. Fertil Steril 2001; 76:172S–3S. 28. Steinman G. Mechanisms of twinning. IV. Sex preference and lactation. J Reprod Med 2001;46:1003–7. 29. Menezo Y, Sakkas D. Monozygotic twinning: is it related to apoptosis in the embryo? Hum Reprod 2002;17:247–51. 30. Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril 2000;74:482–6. 31. Wilson M, Hartke K, Kiehl M, Rodgers J, Brabec C, Lyles R. Integration of blastocyst transfer for all patients. Fertil Steril 2002;77:693–6. 32. Wang H, Dasig D, Gebhardt J, Polan ML, Behr B. Granulocytemacrophage colony-stimulating factor: a regulator in preimplantation embryo development and apoptosis? Fertil Steril 2002;77:7S. 33. Behr B, Dasig D, Gebhardt J, Wang H, Yen W, Polan ML. The gap junction gene Connexin 37 is upregulated by low level GM-CSF in mouse preimplantation embryos. Fertil Steril 2002;77:9S.
Vol. 79, No. 3, March 2003
IN VITRO FERTILIZATION
Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer Amin A. Milki, M.D., Sunny H. Jun, M.D., Mary D. Hinckley, M.D., Barry Behr, Ph.D., Linda C. Giudice, M.D., and Lynn M. Westphal, M.D. Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California
Objective: To evaluate the incidence of monozygotic twinning (MZT) in pregnancies conceived after blastocyst transfer compared to cleavage-stage transfer. Design: Retrospective study. Setting: University IVF program. Patient(s): All IVF patients with viable pregnancies conceived during a 4-year period. Intervention(s): Blastocyst transfer or day 3 ET. Main Outcome Measure(s): Incidence of MZT assessed by transvaginal ultrasound. Result(s): There were 11 incidences of MZT in 197 viable pregnancies (5.6%) with blastocyst transfer compared to 7 of 357 viable pregnancies (2%) with day 3 ET. In 10 of 18 pregnancies, MZT was observed in the setting of a higher order multiple gestation (6 of 11 for blastocyst transfer and 4 of 7 for day 3 ET). In the day 3 ET group, assisted hatching or intracytoplasmic sperm injection (ICSI) did not increase MZT (4 of 213, 1.9%) compared to cycles without zona breaching (3 of 144, 2.1%). Similarly, in the blastocyst-transfer group, ICSI did not increase the incidence of MZT (4 of 74, 5.5% for ICSI and 7 of 123, 5.7% for non-ICSI IVF). Conclusion(s): Compared to day 3 ET, blastocyst transfer appears to significantly increase the incidence of gestations with MZT. This information should be taken into account when counseling patients about the pros and cons of extended culture. (Fertil Steril威 2003;79:503– 6. ©2003 by American Society for Reproductive Medicine.) Key Words: Blastocyst transfer, monozygotic twinning, cleavage stage transfer, multiple gestations, IVF
Received July 5, 2002; revised and accepted September 9, 2002. Presented at the 58th Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, October 14 –17, 2002. Reprint requests: Amin A. Milki, M.D., Department of Gynecology and Obstetrics, Stanford University School of Medicine, 300 Pasteur Drive, HH333, Stanford, California 94305 (FAX: 650498-7294; E-mail: [email protected]). 0015-0282/03/$30.00 doi:10.1016/S0015-0282(02) 04754-4
Monozygotic twinning (MZT) occurs in 0.42% of all births (1). An increased incidence of MZT has been reported in pregnancies conceived after ovulation induction (2) and IVF (3–7), especially when the zona pellucida is breached (4, 5, 8 –10). Although blastocyst transfer can be used to prevent higher order multiple gestations by limiting the transfer to two embryos (11–15), several studies have raised the possibility that MZT is more common with blastocyst transfer (8, 16 –18). However, only two published reports in the literature have specifically assessed the incidence of MZT with blastocyst transfer compared to day 3 ET (17, 18). One of these studies involved only nine pregnancies after blastocyst transfer (17). The other study (18), which looked exclusively at intracytoplasmic sperm injection (ICSI) cycles, compared pregnancies resulting
from blastocyst transfer with those conceived with day 3 ET during the study period and the previous 3 years. The purpose of our study is to compare the incidence of MZT in a large series of day 3 and day 5 ET performed in IVF and ICSI cycles completed during the same time period.
MATERIALS AND METHODS We retrospectively analyzed all viable pregnancies conceived in our IVF program since January 1998, when blastocyst transfer was introduced in our center. The incidence of MZT was examined in pregnancies resulting from blastocyst transfer compared to day 3 ET during this time period. The controlled ovarian hyperstimulation protocol consisted of pretreatment with oral 503
contraceptive (OC) pills with overlapping GnRH agonist down-regulation followed by FSH/hMG and hCG or a microdose flare protocol. Oocytes were inseminated conventionally or by ICSI 3– 4 hours after retrieval. For ICSI, oocytes were denuded using a hyaluronidase solution combined with mechanical stripping. Oocytes were then rinsed and those at the metaphase II stage were injected in phosphate-buffered saline (PBS) with 10% supplement serum substitute (SSS) (Irvine Scientific, Santa Ana, CA). Embryos were cultured in groups under mineral oil in 150-L droplets of P1 medium (Irvine Scientific) with 10% SSS at 37°C in a 5% O2, 5% CO2, and 90% N2 environment for 72 hours. In selected cases, assisted hatching was performed before day 3 ET. Embryos for hatching were placed in PBS with 10% SSS. A small hole (⬍20 m) was made using acidified Tyrode’s solution (pH 2.3) in an area of the zona pellucida that was between blastomeres. Embryos were then rinsed and returned to culture until ET. For the blastocyst transfer group, the embryos were moved on day 3 into blastocyst medium (Irvine Scientific) with 10% SSS and cultured for an additional 48 hours before transfer. All transfers were performed using a Tefcat catheter (Cook Ob/Gyn, Spencer, IN). A viable pregnancy was defined as the presence of cardiac activity confirmed by transvaginal ultrasound at 7 weeks’ gestation. The number of fetuses and gestational sacs was assessed. Monozygotic twinning was diagnosed when more than one fetus with cardiac activity was seen in the same gestational sac. There was no incidence of more gestational sacs than number of embryos transferred. Also, the presence or absence of a dividing amnionic membrane was noted. The findings were reconfirmed with both a repeat ultrasound examination 2 weeks later and follow-up information for the rest of the pregnancy and delivery. Statistical analysis of results was performed using 2 testing. Significance was set at P⬍.05. Institutional review board approval was obtained for review of patient charts.
RESULTS There were 11 incidences of MZT in 197 viable pregnancies (5.6%) with blastocyst transfer compared to 7 of 357 viable pregnancies (2%) with day 3 ET (P⬍.03). Of 127 pregnancies with multiple gestational sacs seen in the entire study population, there were 10 incidences of MZT (6 from blastocyst transfer and 4 from day 3 ET) compared to 8 of 427 pregnancies with a single gestational sac (7.9% vs. 1.9%, P⬍.003). There was no significant difference in the overall multiple gestation rate between the blastocyst transfer group (43 of 197, 22%) and the day 3 ET group (84 of 357, 24%). In the day 3 ET group, the incidence of MZT was not increased by assisted hatching ⫾ ICSI (3 of 155, 1.9%) or ICSI alone (1 of 58, 1.7%) compared to cycles without zona breaching (3 of 144, 2.1%). Similarly, in the blastocyst transfer group, where assisted hatching was not performed, 504
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ICSI did not increase the incidence of MZT (4 of 74, 5.5% for ICSI and 7 of 123, 5.7% for non-ICSI IVF).
DISCUSSION The exact mechanism leading to MZT remains unclear. Factors such as maternal age, family history, race, and parity, which have been associated with dizygotic twinning, do not seem to affect the overall frequency of MZT, which has been reported at 0.42% of all births in the general population (1). It is likely that this incidence would be higher if early first trimester pregnancies were examined. Of the 11 MZ twins reported in our blastocyst transfer pregnancies, 5 spontaneously resolved. Still, the remaining incidence of 3% (6 of 197) represents a sevenfold increase over the natural occurrence of MZT. Ideally, to account for dichorionic MZ twins, the incidence of MZT with IVF should be assessed by looking at transfers of a single embryo leading to a multiple pregnancy. Therefore, our method of identifying MZ twins may underestimate the actual occurrence of this problem by looking only at monochorionic MZ twins. However, these pregnancies are of more clinical relevance due to their known increased perinatal morbidity and mortality. In the context of infertility treatment, Edwards et al. (3) observed a higher than expected frequency of MZ splitting after IVF (1.3%). Derom et al. (2) found a 1.2% frequency of MZ splitting in patients exposed to ovulation induction, which was significantly higher than the expected frequency with spontaneous ovulation. They postulated that in IVF cycles, it is the ovulation induction itself, rather than in vitro conditions, which predisposes to zygotic division or to enhanced survival of MZ twins. Since these early studies, several investigators have reported an increased incidence of MZT in IVF pregnancies (4 –7, 9, 19 –22). Most of these reports have shown that MZT occurred more frequently when the zona pellucida was manipulated to perform assisted hatching, subzonal insemination (SUZI), or ICSI (4, 5, 7–9, 20, 23). These zona breaching techniques result in artificial openings that may interfere with the natural process of hatching. Depending on the actual size and number of these artificial gaps, the embryo may possibly herniate following the path of least resistance and result in a division of the inner cell mass, leading to the formation of identical twins. However, in a recent study, Blickstein et al. (21) showed no increase in MZT with ICSI. Similarly, Sills et al. (6) and Schachter et al. (22) reported no difference in the incidence of MZT in ICSI and assisted hatching cycles compared to IVF without zona breaching. Adding further support to the findings from the latter three studies, our own data did not show a difference in the frequency of MZT in pregnancies derived from ICSI or assisted hatching compared to conventional IVF. Further Vol. 79, No. 3, March 2003
studies of larger samples are still needed to shed light on the association of MZT with zona manipulation. Our study shows that MZT occurs more frequently in pregnancies conceived after blastocyst transfer compared to day 3 ET with an incidence of 5.6% and 2%, respectively. These findings add pertinent information to the literature comparing blastocyst with cleavage-stage transfer in an area that has not been adequately addressed. Initial data presented in an abstract by Rijnders et al. (24) suggested that blastocyst transfer was associated with a higher incidence of MZT compared to cleavage-stage transfer. However, the literature contains only one large published study by da Costa et al. (18), which examined the frequency of MZT in 129 day 5 ET compared to day 3 ET controls, all in ICSI cycles. The data from our study confirm that the increased MZT seen with blastocyst transfer compared to day 3 ET in ICSI cycles, as reported by da Costa et al., is also found in the general IVF population irrespective of whether ICSI was performed. Interestingly, we have found a 7.9% incidence of monozygotic splitting in pregnancies where two or more gestational sacs were present compared to 1.9% in pregnancies with a single gestational sac. This higher rate of MZT in the setting of multiple gestations confirms previous reports. Derom et al. (2) observed that the frequency of splitting was significantly higher in the triplets than in the twins conceived after ovulation induction. Similarly, Wenstrom et al. (19), who reported a 3.2% incidence of MZT in assisted reproduction pregnancies, showed a 9.8% monochorionicity rate in the multiple gestation patients. The overall incidence of multiple gestations in our study population was similar for both day 3 and day 5 ET and is unlikely to be a source of bias accounting for the higher rate of MZT encountered with blastocyst transfer. Herniation of the blastocyst through a less flexible zona, hardened by prolonged in vitro culture, has been proposed as a possible mechanism leading to MZT (4). However, Frankfurter et al. (25) showed a 1.2% MZT rate with zona free (pronase digested) blastocyst transfer, which was not significantly different from the rate seen with zona intact blastocyst transfer in their patients, suggesting that other etiologies must be present. Fertilization in vitro has been associated with a duplication of the inner cell mass (ICM). Chida (26) found that 3.1% of mouse blastocysts fertilized in vitro have a double ICM compared to 0.6% of in vivo fertilized blastocysts. Meintjes et al. (27) reported triplets after the transfer of two blastocysts with one showing two distinct ICMs. The depressed calcium level in a free blastocyst before implantation compared to that found in the endometrium in animal studies has been proposed by Steinman (28) as a mechanism for increased MZT by weakening ICM intercellular bonding and predisposing the embryo to division. This researcher suggests that in vitro incubation for 5 days rather FERTILITY & STERILITY威
than 3 days increases MZT by prolonging the exposure to lower calcium concentrations. However, it would have to be shown that embryos transferred on day 5 implant later than those transferred on day 3 for this theory to be valid. Recently, Menezo and Sakkas (29) proposed a possible explanation for the phenomenon of MZT in blastocyst transfer. They mention that they had no incidence of MZT in more than 800 deliveries when co-culture was used to obtain blastocysts. They believe that media used to grow blastocysts without co-culture may lead to an overstimulation of apoptosis through free radical formation induced by excessive glucose levels. Linear polarization of apoptotic cells in the ICM could lead to splitting during or before the hatching process. They suggest that co-culture using feeder cells may provide free radical scavengers more efficiently than current blastocyst culture media, which could account for the absence of MZT in their patients. In our study, blastocyst medium (Irvine Scientific), which contains 6 mM of glucose, was used. Other reports in the literature suggest that MZT does occur with different blastocyst media with lower glucose concentrations such as S2 or G2.2 (IVF Science, Gothenburg, Sweden). Behr et al. (16) in a multicenter study reported several occurrences of MZT with the use of S2 medium. Da Costa et al. (18) also used S2 for extended culture in their study showing increased MZT with blastocyst transfer. Schoolcraft and Gardner (30) reported two triplet pregnancies resulting from MZT in 89 ongoing pregnancies with blastocyst transfer using G2.2 medium in oocyte donation cycles, whereas Wilson et al. (31), using S2 or G2.2, found that 11 of 12 triplet births from day 5 ET were due to MZT, representing 3.7% of ongoing pregnancies. The true incidence of MZ splitting is very likely to be higher in the latter two studies, as MZT leading to twin gestations was not addressed. On the basis of some recent studies, an alternative explanation, which may account for the absence of MZT when co-culture is used to grow blastocysts, is the production of cytokines or growth factors by mitotically active cells rather than the presence of free radical scavengers. Free radical scavengers like glutathione or taurine are added to many culture media. In particular, high doses of glutathione are present in the blastocyst medium used in our study. Wang et al. (32) demonstrated that in the presence of the growth factor GM-CSF, mouse blastocysts had significantly less apoptotic cells compared to blastocysts grown in standard culture conditions. Behr et al. (33) also demonstrated that the presence of GM-CSF in the culture medium up-regulated the expression of Connexin-37, an integral tight junction protein. Therefore, it is possible that culture media devoid of growth factors or cytokines may impose metabolic stress on embryos that is exacerbated by extended culture. This may translate into higher rates of MZT due in part to increased apoptosis and less cell– cell adhesion. However, caution should be exercised in extrapolating the results from these 505
mouse studies. The data need to be confirmed in human embryos. In conclusion, our study shows that MZT is more common with blastocyst transfer compared to cleavage-stage transfer. Although blastocyst transfer allows the selection of the most viable embryos and accordingly, offers the potential to limit the number of embryos transferred, the reported increase in MZT has to be weighed against the ability of blastocyst transfer to reduce fraternal high order multiple births. The findings from our study should be considered when counseling patients about risks and benefits of extended culture. References 1. Bulmer MG. The biology of twinning in man. Oxford: Clarendon Press, 1970. 2. Derom C, Vlietinck R, Derom R, Van den Berghe H, Thiery M. Increased monozygotic twinning rate after ovulation induction. Lancet 1987;1:1236 –8. 3. Edwards RG, Mettler LE, Walters 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 the human is associated with the 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. 6. Sills ES, Moomjy M, Zaninovic N, Veeck LL, McGee M, Palermo GD, et al. Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF. Hum Reprod 2000;15:890 –5. 7. Abusheikha N, Salha O, Sharma V, Brinsden P. Monozygotic twinning and IVF/ICSI treatment: a report of 11 cases and review of the literature. Hum Reprod Update 2000;6:396 –403. 8. Tarlatzis BC, Qublan HS, Sanopoulou T, Zepiridis L, Grimbizis G, Bontis J. Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer. Fertil Steril 2002;72:196 –8. 9. Saito H, Tsutsumi O, Noda Y, Ibuki Y, Hiroi M. Do assisted reproductive technologies have effects on the demography of monozygotic twinning? Fertil Steril 2000;74:178 –9. 10. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer P, 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. 11. Gardner DK, Schoolcraft WB, Wagley L, Schenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998;13:3434 –40. 12. Milki AA, Fisch JD, Behr B. Two blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared to three blastocyst transfer. Fertil Steril 1999;72:225–8. 13. Karaki RZ, Samarraie SS, Younis NA, Lahloub TM, Ibrahim MH. Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome. Fertil Steril 2002;77:114 –8.
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