- Email: [email protected]
Monozygotic twinning is not associated with zona pellucida micromanipulation procedures but increases with high-order multiple pregnancies
FERTILITY AND STERILITY威 VOL. 82, NO. 2, AUGUST 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Monozygotic twinning is not associated with zona pellucida micromanipulation procedures but increases with high-order multiple pregnancies Between January 1, 1995 and December 31, 2001, 5,310 cycles were performed in the IVF Unit, Sheba Medical Center, Israel, resulting in 1,066 clinical pregnancies. There was no difference in the rate of pregnancies containing monozygotic twins after zona pellucida micromanipulation procedures (0.9%) compared to conventional insemination (1.0%) (6/677 vs. 4/389). (Fertil Steril威 2004;82:500 –1. ©2004 by American Society for Reproductive Medicine.) Monozygotic multiple gestation is of great concern because of an associated increase in neonatal morbidity and mortality. Perinatal mortality risk among monozygotic twins (MZT) has been reported to be two to three times higher than the risk among dizygotic twins. The MZT results from the division of a single fertilized ovum into two genetically identical embryos, and is thought to occur in 0.42% of all deliveries (1). Experimental studies using animal models have called attention to the zona pellucida (ZP) as an important factor in the twinning equation. Zona drilling in mouse blastocyst has been proposed to facilitate twinning (2). Although tropho-ectoderm cells protrude through a small opening created in the ZP, some of them may break off to form MZT. This evidence led to concern that new assisted reproductive technology (ART) involving ZP micromanipulation, such as intracytoplasmic sperm injection (ICSI) and assisted hatching (AHA) may increase the incidence of MZT. Previous studies evaluating ZP micromanipulation as a risk factor to MZT are contradicting (3, 4). The aim of this study was to evaluate the potential association between ZP micromanipulation (ICSI or AHA) and MZT in IVF treatment. The computerized data for all IVF cycles performed between January 1, 1995, and December 31, 2001, at the IVF Unit of the Chaim Sheba Medical Center in Israel were analyzed. The protocol of ovulation induction, oocytes retrieval, culture, fertilization, embryo culture, and transfer were carried out as previously described (5). Approximately 85% of the patients underwent the long protocol of GnRH analog (GnRH-a) administered either in the midluteal or early follicular phase of the menstrual cycle followed by gonadotropin administration after verification of complete ovarian suppression. The remaining 15% of the patients were treated with ovarian stimulation using the flare-up protocol (the GnRH-a was used from day 1 of the menstrual cycle) and stimulation with gonadotropin was started on day 3. During the study period, in our unit, only a few embryos were cultured in vitro to the blastocyst stage; none of them ended in MZT. Therefore, they were not included in our statistics.
Received July 14, 2003; revised and accepted February 26, 2004. Presented at the 58th Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, October 12–17, 2002. Reprint requests: Shai E. Elizur, M.D., IVF Unit, The Chaim Sheba Medical Center, Tel Hashomer 52621, Israel (FAX: 972-3534-1589; E-mail: [email protected]). 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 02.106
500
All clinical pregnancies achieved after IVF-ET cycles between January 1, 1995 and December 31, 2001, were included in the study. The MZT were identified by transvaginal sonography performed 4 weeks after ET when the number of embryos exceeded the number of gestational sacs. A multivariate analysis was performed to define the impact of different factors on the incidence of MZT. Variables evaluated were women’s age, day 3 FSH value during the IVF cycle, ovarian stimulation length and the amount of gonadotropins used, E2 value on the day of hCG injection, the number of oocytes retrieved and fertilized, and the number of embryos transferred. For statistical analysis, the 2 test, Fisher’s exact test, and Student’s t-test were applied as appropriate. Statistical significance was defined as P⬍.05. A total of 5,310 cycles were performed resulting in 1,066 clinical pregnancies. Of these pregnancies, 62% (663/1,066) were singleton pregnancies, 27% (291/1,066) were twins, 9.5% (101/1,066) triplets, and 1.3% (14/1,066) included more than three fetuses. Of all pregnancies, 677 (63.5%) were the result of ZP micromanipulation procedures (581 ICSI cycles, 68 ICSI with AHA, and 28 AHA without ICSI). A total of 10 MZT (0.9%) were identified. There was no difference in the rate of pregnancies with MZT after ZP micromanipulation procedures (0.9%) compared to conventional insemination (1.0%) (6/677 vs. 4/389, P ⫽ not significant [NS]). Calculating the rate of embryos that divided into MZT, 0.25% (6/2,363) of the embryos in the ZP micromanipulation group ended in MZT compared to 0.28% (4/1,437) of the embryos in the control group (not
TABLE 1 The incidence of monozygotic twins (MZT) after zona pellucida micromanipulation procedures. Zona pellucida manipulation (⫹)
Embryos Pregnancies
Zona pellucida manipulation (⫺)
Total
MZT (%) (n ⫽ 6)
Total
MZT (%) (n ⫽ 4)
2,363 677
0.25 0.9
1,437 389
0.28 1.0
Note: Zona pellucida manipulation includes ICSI and assisted hatching. P value nonsignificant. Elizur. MZT in IVF.
Fertil Steril 2004.
statistically significant; Table 1). Of the high-order multiple pregnancies (triplet or more), 5.2% (6/115) contained an MZT. Of the 10 cases of MZT identified, 4 were twin pregnancies, 3 were part of a triplet pregnancy, and 3 were part of a quadruplet pregnancy. Of the four twin pregnancies, two ended in a delivery of twins, one ended in a singleton delivery due to spontaneous abortion of one embryo, and in one case both embryos were spontaneously aborted in the first trimester. One case of a triplet pregnancy ended in triplet delivery and two cases ended in a singleton delivery, one due to elective reduction of the MZT, and the other due to spontaneous abortion of the MZT. Of the quadruplet pregnancies containing MZT, one ended in a triplet delivery due to spontaneous abortion of one embryo, one ended in delivery of dizygotic twins due to elective reduction of the MZT, and one ended in a singleton delivery due to spontaneous abortion of three embryos. None of the analyzed variables were associated with MZT. According to our findings ZP micromanipulation procedures, such as ICSI or AHA, are not risk factors for MZT after IVF-ET treatments. The estimated incidence of MZT per embryo transferred is 0.39% (15/3,800). This incidence is nearly the same as in natural cycles (0.42% per delivery). According to this calculation the high incidence of MZT in IVF-ET may well be due to the higher number of embryos transferred into the uterus compared with natural cycles. A major obstacle in trying to evaluate the impact of ZP micromanipulation procedures on MZT is the overall low incidence of MZT. Because MZT is a relatively rare condition, a very large sample is needed to achieve satisfactory statistical power (more than 10,000 cases). Obviously this is very difficult to achieve in a single center. Therefore, it is necessary to combine as many studies as possible in assessing rare conditions such as MZT. The importance of our study is in being one of the largest studies ever
FERTILITY & STERILITY威
published regarding MZT in ART, combining data already published. In addition, because our data are computerized, reliability and accuracy are ensured. Recent studies (6) emphasized embryo culture in vitro to the blastocyst stage as a possible risk factor in MZT. In our study, few embryos were cultured in vitro to the blastocyst stage and none of them ended in MZT. Therefore, our data cannot assist in clarifying this issue. In our study, 5.2% of the high-order multiple pregnancies (triplet, quadruplet) contained an MZT. The high rate of MZT in high-order multiple pregnancies was also shown by Schachter et al. (3). These complicated multiple pregnancies present a dilemma regarding how to manage such high-risk pregnancies. In our study there were two cases of MZT reduction, one in a triplet pregnancy and the other in a quadruplet pregnancy; both resulted in the delivery of live newborns. Reduction of the MZT seems to be the preferred method of managing these pregnancies due to the high risk of twin-to-twin transfusion syndrome and the overall poor outcome of these pregnancies. In conclusion, according to our findings ZP manipulation procedures are not associated with an increased incidence of MZT in IVF-ET treatment cycles. Because of the low incidence of MZT, additional large studies are needed to improve our understanding of the causes for increased incidence of MZT in IVF-ET treatments. Shai E. Elizur, M.D. Jacob Levron, M.D. Alon Shrim, M.D. Eyal Sivan, M.D. Jehoshua Dor, M.D. Adrian Shulman, M.D. IVF Unit, Sheba Medical Center, Tel Hashomer, affiliated with Sackler Medical School, Tel Aviv University, Israel
References 1. 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. 2. Talansky BE, Gordon JW. Cleavage characteristics of mouse embryos inseminated and cultured after zona pellucida drilling. Gamete Res 1988;21:277–87. 3. 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. 4. 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;77:196 –8. 5. Dor J, Ben-Shlomo I, Levran D, Rudak E, Yunish M, Mashiach S. The relative success of gonadotropin-releasing hormone analogue, clomiphene citrate, and gonadotropin in 1,099 cycles of in vitro fertilization. Fertil Steril 1992;58:986 –90. 6. Da Costa AA, 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.
501
Monozygotic twinning is not associated with zona pellucida micromanipulation procedures but increases with high-order multiple pregnancies Between January 1, 1995 and December 31, 2001, 5,310 cycles were performed in the IVF Unit, Sheba Medical Center, Israel, resulting in 1,066 clinical pregnancies. There was no difference in the rate of pregnancies containing monozygotic twins after zona pellucida micromanipulation procedures (0.9%) compared to conventional insemination (1.0%) (6/677 vs. 4/389). (Fertil Steril威 2004;82:500 –1. ©2004 by American Society for Reproductive Medicine.) Monozygotic multiple gestation is of great concern because of an associated increase in neonatal morbidity and mortality. Perinatal mortality risk among monozygotic twins (MZT) has been reported to be two to three times higher than the risk among dizygotic twins. The MZT results from the division of a single fertilized ovum into two genetically identical embryos, and is thought to occur in 0.42% of all deliveries (1). Experimental studies using animal models have called attention to the zona pellucida (ZP) as an important factor in the twinning equation. Zona drilling in mouse blastocyst has been proposed to facilitate twinning (2). Although tropho-ectoderm cells protrude through a small opening created in the ZP, some of them may break off to form MZT. This evidence led to concern that new assisted reproductive technology (ART) involving ZP micromanipulation, such as intracytoplasmic sperm injection (ICSI) and assisted hatching (AHA) may increase the incidence of MZT. Previous studies evaluating ZP micromanipulation as a risk factor to MZT are contradicting (3, 4). The aim of this study was to evaluate the potential association between ZP micromanipulation (ICSI or AHA) and MZT in IVF treatment. The computerized data for all IVF cycles performed between January 1, 1995, and December 31, 2001, at the IVF Unit of the Chaim Sheba Medical Center in Israel were analyzed. The protocol of ovulation induction, oocytes retrieval, culture, fertilization, embryo culture, and transfer were carried out as previously described (5). Approximately 85% of the patients underwent the long protocol of GnRH analog (GnRH-a) administered either in the midluteal or early follicular phase of the menstrual cycle followed by gonadotropin administration after verification of complete ovarian suppression. The remaining 15% of the patients were treated with ovarian stimulation using the flare-up protocol (the GnRH-a was used from day 1 of the menstrual cycle) and stimulation with gonadotropin was started on day 3. During the study period, in our unit, only a few embryos were cultured in vitro to the blastocyst stage; none of them ended in MZT. Therefore, they were not included in our statistics.
Received July 14, 2003; revised and accepted February 26, 2004. Presented at the 58th Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, October 12–17, 2002. Reprint requests: Shai E. Elizur, M.D., IVF Unit, The Chaim Sheba Medical Center, Tel Hashomer 52621, Israel (FAX: 972-3534-1589; E-mail: [email protected]). 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 02.106
500
All clinical pregnancies achieved after IVF-ET cycles between January 1, 1995 and December 31, 2001, were included in the study. The MZT were identified by transvaginal sonography performed 4 weeks after ET when the number of embryos exceeded the number of gestational sacs. A multivariate analysis was performed to define the impact of different factors on the incidence of MZT. Variables evaluated were women’s age, day 3 FSH value during the IVF cycle, ovarian stimulation length and the amount of gonadotropins used, E2 value on the day of hCG injection, the number of oocytes retrieved and fertilized, and the number of embryos transferred. For statistical analysis, the 2 test, Fisher’s exact test, and Student’s t-test were applied as appropriate. Statistical significance was defined as P⬍.05. A total of 5,310 cycles were performed resulting in 1,066 clinical pregnancies. Of these pregnancies, 62% (663/1,066) were singleton pregnancies, 27% (291/1,066) were twins, 9.5% (101/1,066) triplets, and 1.3% (14/1,066) included more than three fetuses. Of all pregnancies, 677 (63.5%) were the result of ZP micromanipulation procedures (581 ICSI cycles, 68 ICSI with AHA, and 28 AHA without ICSI). A total of 10 MZT (0.9%) were identified. There was no difference in the rate of pregnancies with MZT after ZP micromanipulation procedures (0.9%) compared to conventional insemination (1.0%) (6/677 vs. 4/389, P ⫽ not significant [NS]). Calculating the rate of embryos that divided into MZT, 0.25% (6/2,363) of the embryos in the ZP micromanipulation group ended in MZT compared to 0.28% (4/1,437) of the embryos in the control group (not
TABLE 1 The incidence of monozygotic twins (MZT) after zona pellucida micromanipulation procedures. Zona pellucida manipulation (⫹)
Embryos Pregnancies
Zona pellucida manipulation (⫺)
Total
MZT (%) (n ⫽ 6)
Total
MZT (%) (n ⫽ 4)
2,363 677
0.25 0.9
1,437 389
0.28 1.0
Note: Zona pellucida manipulation includes ICSI and assisted hatching. P value nonsignificant. Elizur. MZT in IVF.
Fertil Steril 2004.
statistically significant; Table 1). Of the high-order multiple pregnancies (triplet or more), 5.2% (6/115) contained an MZT. Of the 10 cases of MZT identified, 4 were twin pregnancies, 3 were part of a triplet pregnancy, and 3 were part of a quadruplet pregnancy. Of the four twin pregnancies, two ended in a delivery of twins, one ended in a singleton delivery due to spontaneous abortion of one embryo, and in one case both embryos were spontaneously aborted in the first trimester. One case of a triplet pregnancy ended in triplet delivery and two cases ended in a singleton delivery, one due to elective reduction of the MZT, and the other due to spontaneous abortion of the MZT. Of the quadruplet pregnancies containing MZT, one ended in a triplet delivery due to spontaneous abortion of one embryo, one ended in delivery of dizygotic twins due to elective reduction of the MZT, and one ended in a singleton delivery due to spontaneous abortion of three embryos. None of the analyzed variables were associated with MZT. According to our findings ZP micromanipulation procedures, such as ICSI or AHA, are not risk factors for MZT after IVF-ET treatments. The estimated incidence of MZT per embryo transferred is 0.39% (15/3,800). This incidence is nearly the same as in natural cycles (0.42% per delivery). According to this calculation the high incidence of MZT in IVF-ET may well be due to the higher number of embryos transferred into the uterus compared with natural cycles. A major obstacle in trying to evaluate the impact of ZP micromanipulation procedures on MZT is the overall low incidence of MZT. Because MZT is a relatively rare condition, a very large sample is needed to achieve satisfactory statistical power (more than 10,000 cases). Obviously this is very difficult to achieve in a single center. Therefore, it is necessary to combine as many studies as possible in assessing rare conditions such as MZT. The importance of our study is in being one of the largest studies ever
FERTILITY & STERILITY威
published regarding MZT in ART, combining data already published. In addition, because our data are computerized, reliability and accuracy are ensured. Recent studies (6) emphasized embryo culture in vitro to the blastocyst stage as a possible risk factor in MZT. In our study, few embryos were cultured in vitro to the blastocyst stage and none of them ended in MZT. Therefore, our data cannot assist in clarifying this issue. In our study, 5.2% of the high-order multiple pregnancies (triplet, quadruplet) contained an MZT. The high rate of MZT in high-order multiple pregnancies was also shown by Schachter et al. (3). These complicated multiple pregnancies present a dilemma regarding how to manage such high-risk pregnancies. In our study there were two cases of MZT reduction, one in a triplet pregnancy and the other in a quadruplet pregnancy; both resulted in the delivery of live newborns. Reduction of the MZT seems to be the preferred method of managing these pregnancies due to the high risk of twin-to-twin transfusion syndrome and the overall poor outcome of these pregnancies. In conclusion, according to our findings ZP manipulation procedures are not associated with an increased incidence of MZT in IVF-ET treatment cycles. Because of the low incidence of MZT, additional large studies are needed to improve our understanding of the causes for increased incidence of MZT in IVF-ET treatments. Shai E. Elizur, M.D. Jacob Levron, M.D. Alon Shrim, M.D. Eyal Sivan, M.D. Jehoshua Dor, M.D. Adrian Shulman, M.D. IVF Unit, Sheba Medical Center, Tel Hashomer, affiliated with Sackler Medical School, Tel Aviv University, Israel
References 1. 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. 2. Talansky BE, Gordon JW. Cleavage characteristics of mouse embryos inseminated and cultured after zona pellucida drilling. Gamete Res 1988;21:277–87. 3. 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. 4. 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;77:196 –8. 5. Dor J, Ben-Shlomo I, Levran D, Rudak E, Yunish M, Mashiach S. The relative success of gonadotropin-releasing hormone analogue, clomiphene citrate, and gonadotropin in 1,099 cycles of in vitro fertilization. Fertil Steril 1992;58:986 –90. 6. Da Costa AA, 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.
501