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intracytoplasmic sperm injection treatment is not related to advanced maternal age, intracytoplasmic sperm injection, assisted hatching, or blastocyst transfer
Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 324e329
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Original Article
Monozygotic twinning after in vitro fertilization/intracytoplasmic sperm injection treatment is not related to advanced maternal age, intracytoplasmic sperm injection, assisted hatching, or blastocyst transfer Dennis Wu, Shang-Yu Huang, Hsien-Ming Wu, Chun-Kai Chen, Yung-Kuei Soong, Hong-Yuan Huang* Department of Obstetrics and Gynecology, Chang-Gung Memorial Hospital and Medical College, Taoyuan, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 11 September 2013
Objective: To evaluate the effect of assisted reproductive techniques on the incidence of monozygotic twins (MZT) and the associated pregnancy outcomes. Materials and methods: This was a retrospective study of all in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles with MZT pregnancies in our center from January 2001 to December 2011. The diagnosis of MZT pregnancies with their respective placental configurations was based on the results of ultrasonographic examinations performed during either the first or second trimester. The treatment characteristics and outcomes of each IVF cycle were recorded and stored in a computer database. Results: A total of 17 cycles with MZT pregnancies were identified, resulting in an overall incidence of MZT of 1.3%. The incidence of MZT for women aged <35 years and 35 years were 1.5% and 0.8%, respectively (p ¼ 0.319). The incidence was not significantly different between ICSI and non-ICSI cycles (1.4% vs. 1.0%; p ¼ 0.620). In addition, the incidence was not increased in the assisted hatching (AH) group compared to those without AH (0.9% vs. 2.1%; p ¼ 0.103). Finally, cycles with embryo transfer at the blastocyst stage had an MZT incidence that was not significantly different from those transferred at the cleavage stage (1.4% vs. 1.3%, respectively; p ¼ 1.000). The incidence of each type of chorionicity, dichorionicediamniotic, monochorionicediamniotic, and monochorionicemonoamniotic, was 33.3%, 46.7%, and 20.0%, respectively. A total of 11 of 39 (28%) monozygotic babies and 16 of 19 (84%) coexisting heterozygotic babies were born alive. Conclusion: Until definite conclusions are drawn from larger trials, patients receiving IVF should not be overly concerned about the increase in MZT risk when proceeding to various assisted reproductive procedures (i.e., ICSI, AH, and blastocyst transfer). However, there is some evidence that the incidence of monochorionicemonoamniotic twins may be significantly increased after IVF/ICSI cycles. Patients should be informed about the possible obstetric complications regarding this rare type of MZT. Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: assisted hatching in vitro fertilization intracytoplasmic sperm injection monozygotic twinning
Introduction Monozygotic twinning (MZT) occurs rarely in natural conception, with an estimated overall incidence of 0.4e0.45% [1,2]. Unlike dizygotic twinning, in which the incidence varies among different races, monozygotic twinning is evenly distributed in all populations * Corresponding author. Department of Obstetrics and Gynecology, Chang-Gung Memorial Hospital, 5, Fu-Hsing Street, Kwei-Shan, Taoyuan 333, Taiwan. E-mail address: [email protected] (H.-Y. Huang).
around the world [3]. The use of assisted reproductive techniques (ART) has been suggested to be associated with an increased occurrence of MZT, which is approximately 1.5% [1,4,5]. However, the actual ART-related incidence is obscured by multiple embryo transfer and early embryonic demise. Several previously proposed factors contributing to the increased incidence of MZT in ART include ovarian stimulation, intracytoplasmic sperm injection (ICSI), assisted hatching (AH), and embryo culture. However, the exact mechanism that leads to the association between ART and MZT is still unclear and needs further investigation.
http://dx.doi.org/10.1016/j.tjog.2014.07.001 1028-4559/Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
D. Wu et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 324e329
MZT occurs when a single zygote divides into two separate embryos. If the cleavage of the zygote occurs later than the 4th day after fertilization, monochorionic twins (with or without the sharing of placenta), monoamniotic twins, or even conjoined twins may develop. MZT is associated with several significant obstetric complications, including preterm labor, intrauterine growth restriction, and fetal death. Twinetwin transfusion syndrome (TTTS) occurs in approximately 9e15% of monochorionic twins [6,7], and the neurologic sequelae of the surviving twins generate further concerns. With the recent trend of the increasing use of ART, preconception counseling about the possibility of monozygotic twins becomes crucial prior to an ART protocol. Furthermore, early detection of the zygosity by transvaginal sonography in the first or early second trimester should be routinely performed in clinical practice. In this retrospective study, we report all MZT gestations from in vitro fertilization (IVF) cycles performed between 2000 and 2011 in our fertility center. The available laboratory data, treatment protocols, and pregnancy outcomes were reviewed accordingly. The objective was to identify any association between the ART procedures and the incidence of MZT pregnancy. Materials and methods This study consists of all IVF/ICSI cycles with embryo transfer performed in our center from January 2001 to December 2011. Patients were assigned to either the long protocol (downregulation protocol) using a GnRH agonist (Lupron; Takeda, Osaka, Japan) or the GnRH antagonist (Cetrotide; Serono, Geneva, Switzerland) protocol according to their fertility evaluations and/or previous treatment responses. Both protocols combined the use of recombinant gonadotropin injections (Gonal-F; Industria Farmaceutica, Serono, Italy; or Puregon; N.V. Oganon, Oss, The Netherlands) initiating on Day 2e3 of the stimulation cycle. The dosage of recombinant gonadotropin was adjusted according to serum estradiol (E2) levels and follicular growth monitored by transvaginal ultrasonography. Final oocyte maturation was triggered by 10,000 IU of urinary human chorionic gonadotropin (Pregnyl; MSD, Haarlem, The Netherlands) when there were two or more follicles larger than 1.7 cm. Oocyte retrieval was performed 36 hours after human chorionic gonadotropin administration under the guidance of transvaginal ultrasonography. Progesterone supplementation began on the day of oocyte retrieval and continued until a fetal heartbeat was confirmed. After serial washing, each oocyte recovered was maintained at 37 C in a separate drop of fertilization medium (Cook IVF, Brisbane, QL, Australia) equilibrated with 6% CO2 in air. Insemination was carried out either through the conventional IVF procedure or ICSI based on the results of semen analysis. The zygotes were examined for the presence of pronuclei and polar bodies at 18e21 hours after insemination. Zygotes with two pronuclei were cultured at 37 C in a separate drop of cleavage culture medium equilibrated with 6% CO2 in air until Day 3. Embryos were assessed daily on developmental rate and morphologic appearances to determine the length of culture. If embryos were cultured beyond Day 3, blastocyst culture medium (Cook IVF) was used. The best quality embryos were selected for transfer. These embryos were selectively treated with AH on the day of embryo transfer. We used laser quarter thinning of the zona pellucida (ZP), as described previously [8,9], as our method of AH. Routine transvaginal ultrasonographic examinations were arranged for the 6th gestational week or 7th gestational week for patients with a positive urinary pregnancy test result. The following diagnostic criteria were used to determine monozygotic pregnancies: the number of fetal poles exceeded the number of embryos
325
transferred or more than one fetal pole was identified in a single gestational sac. Follow-up obstetric ultrasonographic examinations were arranged at 10e14 weeks of gestation to confirm fetal viability, chorionicity, and fetal sex. Chorionicity was determined by the presence of either a “T-sign” (which signifies monochorionic twins) at the intertwin membrane-placental junction or a “l-sign” (which suggests dichorionic twins) [10], whereas fetal sex was identified by checking for the sagittal sign, as described previously [11,12]. All ultrasonographic examinations were performed by wellexperienced ultrasonographic technicians, and all images were reviewed by gynecologists to confirm the diagnosis. The treatment characteristics and outcomes of each IVF cycle were recorded and stored in a computer database. Chi-square tests were used to analyze the effect of each proposed risk factor on the incidence of MZT. A p value <0.05 was considered statistically significant. Results The total number of IVF cycles from January 2001 to December 2011 with a positive pregnancy result was 1338, in which 17 pairs of MZT were identified, resulting in an overall incidence of MZT of 1.3% in this study. All 17 pairs of MZT resulted from the transfer of fresh embryos. Only one pair of MZT resulted from the use of donated oocytes, and the rest of the MZT pairs resulted from embryo transfer of autologous oocytes. We did not analyze these two groups separately because the number of MZT pairs resulting from donated oocytes was small. The summary of patient characteristics, treatment protocols, and chorionicities of the 17 MZT pregnancies is shown in Table 1, and the proposed MZT risk factors with their respective incidences of MZT gestation are summarized in Table 2. The mean maternal age of MZT pregnancies was 32.1 years. To analyze the effects of maternal age on the incidence of MZT pregnancy, we compared IVF cycles performed in women aged <35 years with women 35 years. The results showed that the incidence of MZT pregnancy was higher in cycles performed in women aged <35 years, although the difference was not statistically significant (1.5% vs. 0.8%, respectively; p ¼ 0.319). The effect of ICSI on the incidence of MZT pregnancy was analyzed. There were 853 fresh IVF cycles with positive pregnancy results in which ICSI was performed. Twelve ICSI cycles (1.4%) developed MZT gestations. For fresh IVF cycles without ICSI, the incidence of MZT was 1.0%. However, the difference was not statistically significant (p ¼ 0.620). We also examined the effect of AH on the incidence of MZT pregnancy, but the results showed no increase in MZT incidence in the AH group compared to those without AH (0.9% vs. 2.1%). The influence of embryo culture length on the incidence of MZT pregnancy was also reviewed. The incidence of MZT pregnancy in the Day 5 group was 1.4% (2 out of 151), which is not significantly different from the 1.3% (15 out of 1191) incidence in the cleavage stage group. The chorionicity of MZT pregnancies was determined via transvaginal ultrasonographic examinations performed in the first and/or second trimester (Figure 1). Two MZT gestations had undetermined chorionicities due to early fetal demise. Ten (67%) of the MZT pairs with known chorionicities were monochorionic, whereas five (33%) were dichorionic. Of the monochorionic MZT pregnancies, seven (70%) were diamniotic and three (30%) were monoamniotic. The obstetric outcomes of all 17 cases with MZT pregnancies are summarized in Table 3. Fourteen cases resulted in at least one live birth, including those with coexisting heterozygotic infant (s). In six of these cycles, at least one monozygotic baby was born alive. A coexisting viable heterozygotic infant was delivered at the same time in two cases. Only one pair of monochorionicemonoamniotic
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Table 1 Summary of patient characteristics, treatment protocols, and chorionicities. Case no.
Maternal age (y)
Type of infertility
Etiology
IVF protocol
ICSI
AH
Day of ET
No. of embryos transferred
OHSS
Chorionicity
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
33 28 29 32 32 35 28 31 32 33 31 36 37 33 34 30 41
Secondary Primary Primary Primary Primary Secondary Primary Secondary Secondary Primary Primary Primary Primary Primary Secondary Primary Primary
Tubal factor Male factor Male factor Tubal factor Unexplained Tubal factor Tubal factor Ovulatory factor Ovulatory factor Unexplained Combined Male factor Male factor Male factor Tubal factor Ovulatory factor Ovulatory factor
Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Antagonist Antagonist Long agonist Long agonist Antagonist Antagonist Antagonist
þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
3 3 3 3 2 3 2 2 3 2 3 3 3 2 5 4 5
5 6 5 3 2 3 3 3 4 4 3 3 4 2 4 3 3
þ þ
DCDA MCDA MCDA MCDA DCDA DCDA MCMA MCDA MCDA MCMA MCMA Unknown Unknown DCDA DCDA MCDA MCDA
AH ¼ assisted hatching; DCDA ¼ dichorionicediamniotic; ET ¼ embryo transfer; ICSI ¼ intracytoplasmic sperm injection; MCDA ¼ monochorionicediamniotic; MCMA ¼ monochorionicemonoamniotic; OHSS ¼ ovarian hyperstimulation syndrome.
(MCMA) twins survived in this study (Case 11). In particular, one case developed monozygotic triplets, and all three babies were delivered alive (Case 17). In the three cases in which there was no live birth, one had a coexisting singleton and resulted in triplet fetal deaths during the first trimester. The other two cases with only MZT fetuses acquired TTTS; both were monochorionicediamniotic (MCDA) twins and each received an elective termination at 26 weeks and 30 weeks of gestation, respectively. A total of nine cases received an elective fetal reduction, whereas spontaneous fetal loss occurred in six. In summary, a total of 58 fetuses developed from 17 IVF cycles with MZT pregnancies. Thirty-nine were monozygotic and 19 were coexisting singletons or twins. Eleven of 39 (28%) monozygotic babies and 16 of 19 (84%) coexisting heterozygotic fetuses were born alive. Discussion Monozygotic twin pregnancies have an increased risk of obstetric complications and poor pregnancy outcomes. Although it is a rare event, its incidence had been reported in previous studies to increase by as much as four-fold as a result of ART procedures [1,4,5,13,14]. The trend towards late marriage and delaying conception has resulted in the increased practice of ART in many countries worldwide. This phenomenon is even more markedly demonstrated in Taiwan by the increase of total IVF cycles performed from 2001 to 2010 (from 6458 to 11,513, a 78% increase). The percentage of live births resulting from IVF cycles has also increased from 0.8% to 1.8% [15]. Therefore, MZT gestations may become an issue as a growing number of ART practices persist. Unlike dizygotic multiple pregnancies, which can be easily explained by multiple ovulations or multiple embryo implantations, the actual mechanism resulting in the development of Table 2 Summary of proposed monozygotic twinning (MZT) risk factors with their respective MZT incidences. Proposed MZT risk factor
Maternal age > 35 y ICSI Assisted hatching Day-5 transfer
MZT incidence (%)
p
With risk factor
Without risk factor
0.82 1.40 0.94 1.35
1.53 1.02 2.10 1.26
ICSI ¼ intracytoplasmic sperm injection.
0.319 0.620 0.103 1.000
monozygotic multiple pregnancies remains uncertain. It seems obvious that the moment of embryonic cleavage plays an important part in the outcome. However, there is not yet a single theory explaining the mechanism of zygotic splitting that is responsible for the increased incidence of MZT as a result of various assisted reproduction methods. Several aspects of ART techniques have been discussed previously in an attempt to link their associations with MZT incidence, including maternal age, ICSI, AH, PGD, and length of embryo culture. In the current study, the overall incidence of MZT pregnancy was 1.3%, a result comparable to previously reported data, which was approximately three times higher than a rate of 0.4% from natural conceptions [1,16e18]. Previous reports have suggested that advanced maternal age increases the incidence of spontaneous MZT pregnancies [16]. It has been proposed that the average thickness of the ZP gradually decreases with increasing maternal age [19e22], which may alter the normal hatching process, for instance, the splitting of blastocyst during its protrusion due to multiple sites of zona lysis [23]. However, two recent studies reported outcomes that conflict with the concept of advanced maternal age being a risk factor [24,25]. The correlation between zona properties and maternal age was further challenged by two other recent published reports [26,27]. Interestingly, our data also showed a trend towards a higher incidence of MZT pregnancies in women aged <35 years compared to those aged 35 years (1.5% vs. 0.8%; p ¼ 0.319). The use of micromanipulation in ART has been frequently linked with the development of MZT. The proposed mechanism for this link is related to ZP damage that creates gaps, which induce herniation in the blastomere membranes, causing embryo splitting. Previous reports compared MZT incidences between ZP manipulation procedures such as ICSI or AH and conventional IVF. Although some studies found an increased incidence of MZT pregnancy after ZP manipulation [2,23,28e30], many others did not observe this association [14,18,31e34]. In the current study, we also failed to observe significant difference in MZT incidence between IVF with zona manipulation and conventional IVF. The contradictory results between the various studies may be due to the following reasons. First, most of the studies, including the current report, were carried out as retrospective reviews. The significant heterogeneity between these study groups makes it difficult to compare and analyze the results objectively. Furthermore, there were various reported techniques of performing ZP manipulation, and the procedures used in the earlier reports might have
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Fig. 1. Summary of documented chorionicity of monozygotic twin (MZT) pregnancies.
been replaced by newer techniques described in later studies [9]. The question of whether different techniques of micromanipulation procedures can affect the incidence of MZT pregnancy remained to be answered. Finally, there may exist unknown mechanisms during IVF procedures, other than ZP manipulation, that are involved in embryo splitting. The other factor being discussed that may affect the MZT pregnancy incidence is the stage at which the embryo is transferred. With the introduction of sequential culture media, blastocyst transfer has gained increasing popularity over the past decade [35]. It has long been accepted that transferring an embryo at the blastocyst stage enhances implantation rates. In this way, the number of transferred embryos can be reduced to lower the risk of multiple pregnancies without decreasing the pregnancy rate [36]. Nevertheless, many studies have reported an increased risk of MZT pregnancies after a blastocyst transfer compared with a cleavage stage embryo transfer [24,29,32,37e39]. This increase has been explained by several mechanisms, including prolonged culture time [40], culture media composition [41,42], and the experience of laboratory embryologists [37,43]. By contrast, other recent studies did not find an increase in MZT pregnancy rate after blastocyst transfer [44,45]. Our findings also showed no significant difference in MZT risk between the two stages of embryo transfer. Again, many of these studies were small series studies, which resulted in great variations in MZT rates. Further larger trials are required to investigate whether the stage at which the embryo is transferred has an impact on MZT rate.
To evaluate the obstetric outcomes of MZT pregnancies, we discuss not only the effect of IVF procedures on the incidence of MZT pregnancy but also the membrane configuration of each MZT cycle. In the current study, the percentage of each type of chorionicity (dichorionicediamniotic, MCDA, and MCMA) was 33.3%, 46.7%, and 20.0%, respectively. In particular, the incidence of MCMA twins was much higher than those from spontaneous MZT (1e2%) [46]. This finding is similar to the result of a previous study that reported the significantly increased incidence of MCMA twins (37%) after ART treatments [13]. Although the relative occurrence of each type of chorionicity of ART-derived MZT requires further supporting data, the results raise concerns that the obstetric risk may be elevated in women receiving ART procedures. MCMA twins are well known for their high risk of cord entanglement and TTTS. As a consequence, previous reports have emphasized the high perinatal loss rate in MCMA twins (range, 19e44%) [47e50]. Fortunately, the three cases of MCMA twin pregnancies in our study had relatively good outcomes. Two received elective fetal reduction and resulted in live birth deliveries of coexisting heterozygotic twins. One had a coexisting singleton delivered alive with the MCMA twins, which made them the only surviving MCMA twins in this study. By contrast, the only two cases with TTTS had MCDA twins. Both received an elective termination during the early third trimester due to poor fetal conditions. In conclusion, this study represents the first single-center experience that analyzes the relationship between MZT incidence and ART in Taiwan. In addition, the pregnancy outcomes were also
Table 3 Summary of pregnancy outcomes. Case no.
Monozygotic twins/triplets
Coexisting heterozygotic infant (s)
Delivery method
Gestational age (wk)
Birth weight (g)
Sex
1
Fetal reduction 2
Cesarean
34
1540, 2080
F, M
2 3 4 5 6 7 8 9
Expired 2 Expired 2 IUFD 3 Fetal reduction Fetal reduction Fetal reduction Alive 2 Fetal reduction
Fetal reduction 1 Alive 2 0 0 0 0 Alive 2 Alive 2 0 Alive 1
Vaginal Vaginal Vaginal Cesarean Cesarean Cesarean Cesarean Cesarean
30 26 11 31 36 34 32 27
1120/899 695/775 10/5/5 1350/1610 2550, 1865 2020, 1850 2260/2250 1039
F/F Ambiguous Ambiguous M, F F, F M, F M/M F
10 11 12 13 14 15 16 17
Fetal reduction 4 Alive 2 Fetal reduction 2 Alive 1, IUFD 1 Alive 1, IUFD 1 Fetal reduction 2 Fetal reduction 2 Alive 3
Cesarean Cesarean Cesarean Cesarean Cesarean Cesarean Vaginal Cesarean
35 34 40 35 36 36 38 31
2390, 950 1745/1670, 1830 3140 2555, 2375 2750, 2190 2070, 2095 2890 1740/1285/1560
M, M M/M, F M F, F F, M F, M M M/M/M
2, Alive 2 2 2 2
Alive Alive Alive Alive Alive Alive Alive 0
2 1 1 1 1 2 1
IUFD ¼ intrauterine fetal death; TTTS ¼ twinetwin transfusion syndrome.
Complication
At least 1 live birth þ
TTTS TTTS
Maternal pulmonary edema
þ þ þ þ þ þ þ þ þ þ þ þ þ
328
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summarized. Our results concur with previously reported data that the rate of MZT is higher in women receiving ART compared with that of spontaneous MZT. Our study also agreed with recent findings that MZT pregnancies may be associated with younger women. However, we did not find a significant increase in the MZT rate from IVF cycles with micromanipulation or blastocyst transfer. Lastly, the incidence of MCMA twins (which is believed to be the rarest form of MZT) appeared to increase significantly after ART. There are several limitations to this study that must be discussed. Similar to many of the previous studies, the current study was carried out through a retrospective review. In addition, different age groups might receive different ART, which makes objective comparisons difficult. Furthermore, the diagnosis of MZT was based on the findings of the first or second trimester ultrasound, which identified those with a number of fetuses exceeding the number of embryos transferred. The confirmation of MZT through neonatal DNA analysis was not performed in any of the cycles. This fact may lead to the underestimation of MZT, as some MZT might have been misinterpreted as fraternal twins in cases of dichorionicediamniotic twins. Lastly, this study was conducted based on a single center experience, which provided us with a relatively small sample size. The rare occurrence of MZT incidence also resulted in the paucity of cases of interest. Further larger trials are required to draw solid conclusions on this topic. MZT has long been associated with the use of ART. Based on the findings of current study and previous studies, patients receiving IVF should not be overly concerned about the increase in MZT risk when proceeding to various other ART (i.e., ICSI, AH, and blastocyst transfer). However, the incidence of MCMA twins may be significantly increased after IVF/ICSI cycles. We suggest the initiation of national scale multicenter trials to assess the relative risks of each proposed contributing factor of MZT. Through a better understanding of the relationship between ART and MZT, we will be able to reduce the incidence of MZT and thus lower the potential obstetric risks in future IVF cycles. Conflicts of interest The authors have no conflicts of interest relevant to this article. References [1] Derom C, Vlietinck R, Derom R, Van den Berghe H, Thiery M. Increased monozygotic twinning rate after ovulation induction. Lancet 1987;1:1236e8. [2] 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;74:178e9. [3] Bortolus R, Parazzini F, Chatenoud L, Benzi G, Bianchi MM, Marini A. The epidemiology of multiple births. Hum Reprod Update 1999;5:179e87. [4] Edwards RG, Mettler L, Walters DE. Identical twins and in vitro fertilization. J In Vitro Fert Embryo Transf 1986;3:114e7. [5] Wenstrom KD, Syrop CH, Hammitt DG, Van Voorhis BJ. Increased risk of monochorionic twinning associated with assisted reproduction. Fertil Steril 1993;60:510e4. [6] Sebire NJ, Snijders RJ, Hughes K, Sepulveda W, Nicolaides KH. The hidden mortality of monochorionic twin pregnancies. Br J Obstet Gynaecol 1997;104: 1203e7. [7] Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Kirmeyer S, et al. Births: final data for 2005. Natl Vital Stat Rep 2007;56:1e103. [8] Blake DA, Forsberg AS, Johansson BR, Wikland M. Laser zona pellucida thinningdan alternative approach to assisted hatching. Hum Reprod 2001;16: 1959e64. [9] Mantoudis E, Podsiadly BT, Gorgy A, Venkat G, Craft IL. A comparison between quarter, partial and total laser assisted hatching in selected infertility patients. Hum Reprod 2001;16:2182e6. [10] Sepulveda W, Sebire NJ, Hughes K, Odibo A, Nicolaides KH. The lambda sign at 10-14 weeks of gestation as a predictor of chorionicity in twin pregnancies. Ultrasound Obstet Gynecol 1996;7:421e3. [11] Mazza V, Falcinelli C, Paganelli S, Contu G, Mantuano SM, Battafarano SD, et al. Sonographic early fetal gender assignment: a longitudinal study in pregnancies after in vitro fertilization. Ultrasound Obstet Gynecol 2001;17:513e6.
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Does assisted hatching pose a risk for monozygotic twinning in pregnancies conceived through in vitro fertilization? Fertil Steril 2000;74:288e94. [29] Skiadas CC, Missmer SA, Benson CB, Gee RE, Racowsky C. Risk factors associated with pregnancies containing a monochorionic pair following assisted reproductive technologies. Hum Reprod 2008;23:1366e71. [30] 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;77:196e8. [31] Behr B, Fisch JD, Racowsky C, Miller K, Pool TB, Milki AA. Blastocyst-ET and monozygotic twinning. J Assist Reprod Genet 2000;17:349e51. [32] Milki AA, Jun SH, Hinckley MD, Behr B, Giudice LC, Westphal LM. Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer. Fertil Steril 2003;79:503e6. [33] Elizur SE, Levron J, Shrim A, Sivan E, Dor J, Shulman A. Monozygotic twinning is not associated with zona pellucida micromanipulation procedures but increases with high-order multiple pregnancies. Fertil Steril 2004;82:500e1. [34] Yanaihara A, Yorimitsu T, Motoyama H, Watanabe H, Kawamura T. Monozygotic multiple gestation following in vitro fertilization: analysis of seven cases from Japan. J Exp Clin Assist Reprod 2007;4:4. [35] Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998;69:84e8. [36] Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998;13:3434e40. [37] Jain JK, Boostanfar R, Slater CC, Francis MM, Paulson RJ. Monozygotic twins and triplets in association with blastocyst transfer. J Assist Reprod Genet 2004;21:103e7. [38] Wright V, Schieve LA, Vahratian A, Reynolds MA. Monozygotic twinning associated with day 5 embryo transfer in pregnancies conceived after IVF. Hum Reprod 2004;19:1831e6. [39] Vitthala S, Gelbaya TA, Brison DR, Fitzgerald CT, Nardo LG. The risk of monozygotic twins after assisted reproductive technology: a systematic review and meta-analysis. Hum Reprod Update 2009;15:45e55. [40] 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;16:333e6. ne zo YJ, Sakkas D. Monozygotic twinning: is it related to apoptosis in the [41] Me embryo? Hum Reprod 2002;17:247e8.
D. Wu et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 324e329 [42] Cassuto G, Chavrier M, Menezo Y. Culture conditions and not prolonged culture time are responsible for monozygotic twinning in human in vitro fertilization. Fertil Steril 2003;80:462e3. [43] Moayeri SE, Behr B, Lathi RB, Westphal LM, Milki AA. Risk of monozygotic twinning with blastocyst transfer decreases over time: an 8-year experience. Fertil Steril 2007;87:1028e32. [44] Sharara FI, Abdo G. Incidence of monozygotic twins in blastocyst and cleavage stage assisted reproductive technology cycles. Fertil Steril 2010;93:642e5. [45] Papanikolaou EG, Fatemi H, Venetis C, Donoso P, Kolibianakis E, Tournaye H, et al. Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer. Fertil Steril 2010;93: 592e7.
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[46] Hall JG. Twinning. Lancet 2003;362:735e43. [47] Hack KE, Derks JB, Schaap AH, Lopriore E, Elias SG, Arabin B, et al. Perinatal outcome of monoamniotic twin pregnancies. Obstet Gynecol 2009;113: 353e60. [48] Ezra Y, Shveiky D, Ophir E, Nadjari M, Eisenberg VH, Samueloff A, et al. Intensive management and early delivery reduce antenatal mortality in monoamniotic twin pregnancies. Acta Obstet Gynecol Scand 2005;84:432e5. [49] Sau AK, Langford K, Elliott C, Su LL, Maxwell DJ. Monoamniotic twins: what should be the optimal antenatal management? Twin Res 2003;6:270e4. [50] Dias T, Mahsud-Dornan S, Bhide A, Papageorghiou AT, Thilaganathan B. Cord entanglement and perinatal outcome in monoamniotic twin pregnancies. Ultrasound Obstet Gynecol 2010;35:201e4.
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Original Article
Monozygotic twinning after in vitro fertilization/intracytoplasmic sperm injection treatment is not related to advanced maternal age, intracytoplasmic sperm injection, assisted hatching, or blastocyst transfer Dennis Wu, Shang-Yu Huang, Hsien-Ming Wu, Chun-Kai Chen, Yung-Kuei Soong, Hong-Yuan Huang* Department of Obstetrics and Gynecology, Chang-Gung Memorial Hospital and Medical College, Taoyuan, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 11 September 2013
Objective: To evaluate the effect of assisted reproductive techniques on the incidence of monozygotic twins (MZT) and the associated pregnancy outcomes. Materials and methods: This was a retrospective study of all in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles with MZT pregnancies in our center from January 2001 to December 2011. The diagnosis of MZT pregnancies with their respective placental configurations was based on the results of ultrasonographic examinations performed during either the first or second trimester. The treatment characteristics and outcomes of each IVF cycle were recorded and stored in a computer database. Results: A total of 17 cycles with MZT pregnancies were identified, resulting in an overall incidence of MZT of 1.3%. The incidence of MZT for women aged <35 years and 35 years were 1.5% and 0.8%, respectively (p ¼ 0.319). The incidence was not significantly different between ICSI and non-ICSI cycles (1.4% vs. 1.0%; p ¼ 0.620). In addition, the incidence was not increased in the assisted hatching (AH) group compared to those without AH (0.9% vs. 2.1%; p ¼ 0.103). Finally, cycles with embryo transfer at the blastocyst stage had an MZT incidence that was not significantly different from those transferred at the cleavage stage (1.4% vs. 1.3%, respectively; p ¼ 1.000). The incidence of each type of chorionicity, dichorionicediamniotic, monochorionicediamniotic, and monochorionicemonoamniotic, was 33.3%, 46.7%, and 20.0%, respectively. A total of 11 of 39 (28%) monozygotic babies and 16 of 19 (84%) coexisting heterozygotic babies were born alive. Conclusion: Until definite conclusions are drawn from larger trials, patients receiving IVF should not be overly concerned about the increase in MZT risk when proceeding to various assisted reproductive procedures (i.e., ICSI, AH, and blastocyst transfer). However, there is some evidence that the incidence of monochorionicemonoamniotic twins may be significantly increased after IVF/ICSI cycles. Patients should be informed about the possible obstetric complications regarding this rare type of MZT. Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: assisted hatching in vitro fertilization intracytoplasmic sperm injection monozygotic twinning
Introduction Monozygotic twinning (MZT) occurs rarely in natural conception, with an estimated overall incidence of 0.4e0.45% [1,2]. Unlike dizygotic twinning, in which the incidence varies among different races, monozygotic twinning is evenly distributed in all populations * Corresponding author. Department of Obstetrics and Gynecology, Chang-Gung Memorial Hospital, 5, Fu-Hsing Street, Kwei-Shan, Taoyuan 333, Taiwan. E-mail address: [email protected] (H.-Y. Huang).
around the world [3]. The use of assisted reproductive techniques (ART) has been suggested to be associated with an increased occurrence of MZT, which is approximately 1.5% [1,4,5]. However, the actual ART-related incidence is obscured by multiple embryo transfer and early embryonic demise. Several previously proposed factors contributing to the increased incidence of MZT in ART include ovarian stimulation, intracytoplasmic sperm injection (ICSI), assisted hatching (AH), and embryo culture. However, the exact mechanism that leads to the association between ART and MZT is still unclear and needs further investigation.
http://dx.doi.org/10.1016/j.tjog.2014.07.001 1028-4559/Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
D. Wu et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 324e329
MZT occurs when a single zygote divides into two separate embryos. If the cleavage of the zygote occurs later than the 4th day after fertilization, monochorionic twins (with or without the sharing of placenta), monoamniotic twins, or even conjoined twins may develop. MZT is associated with several significant obstetric complications, including preterm labor, intrauterine growth restriction, and fetal death. Twinetwin transfusion syndrome (TTTS) occurs in approximately 9e15% of monochorionic twins [6,7], and the neurologic sequelae of the surviving twins generate further concerns. With the recent trend of the increasing use of ART, preconception counseling about the possibility of monozygotic twins becomes crucial prior to an ART protocol. Furthermore, early detection of the zygosity by transvaginal sonography in the first or early second trimester should be routinely performed in clinical practice. In this retrospective study, we report all MZT gestations from in vitro fertilization (IVF) cycles performed between 2000 and 2011 in our fertility center. The available laboratory data, treatment protocols, and pregnancy outcomes were reviewed accordingly. The objective was to identify any association between the ART procedures and the incidence of MZT pregnancy. Materials and methods This study consists of all IVF/ICSI cycles with embryo transfer performed in our center from January 2001 to December 2011. Patients were assigned to either the long protocol (downregulation protocol) using a GnRH agonist (Lupron; Takeda, Osaka, Japan) or the GnRH antagonist (Cetrotide; Serono, Geneva, Switzerland) protocol according to their fertility evaluations and/or previous treatment responses. Both protocols combined the use of recombinant gonadotropin injections (Gonal-F; Industria Farmaceutica, Serono, Italy; or Puregon; N.V. Oganon, Oss, The Netherlands) initiating on Day 2e3 of the stimulation cycle. The dosage of recombinant gonadotropin was adjusted according to serum estradiol (E2) levels and follicular growth monitored by transvaginal ultrasonography. Final oocyte maturation was triggered by 10,000 IU of urinary human chorionic gonadotropin (Pregnyl; MSD, Haarlem, The Netherlands) when there were two or more follicles larger than 1.7 cm. Oocyte retrieval was performed 36 hours after human chorionic gonadotropin administration under the guidance of transvaginal ultrasonography. Progesterone supplementation began on the day of oocyte retrieval and continued until a fetal heartbeat was confirmed. After serial washing, each oocyte recovered was maintained at 37 C in a separate drop of fertilization medium (Cook IVF, Brisbane, QL, Australia) equilibrated with 6% CO2 in air. Insemination was carried out either through the conventional IVF procedure or ICSI based on the results of semen analysis. The zygotes were examined for the presence of pronuclei and polar bodies at 18e21 hours after insemination. Zygotes with two pronuclei were cultured at 37 C in a separate drop of cleavage culture medium equilibrated with 6% CO2 in air until Day 3. Embryos were assessed daily on developmental rate and morphologic appearances to determine the length of culture. If embryos were cultured beyond Day 3, blastocyst culture medium (Cook IVF) was used. The best quality embryos were selected for transfer. These embryos were selectively treated with AH on the day of embryo transfer. We used laser quarter thinning of the zona pellucida (ZP), as described previously [8,9], as our method of AH. Routine transvaginal ultrasonographic examinations were arranged for the 6th gestational week or 7th gestational week for patients with a positive urinary pregnancy test result. The following diagnostic criteria were used to determine monozygotic pregnancies: the number of fetal poles exceeded the number of embryos
325
transferred or more than one fetal pole was identified in a single gestational sac. Follow-up obstetric ultrasonographic examinations were arranged at 10e14 weeks of gestation to confirm fetal viability, chorionicity, and fetal sex. Chorionicity was determined by the presence of either a “T-sign” (which signifies monochorionic twins) at the intertwin membrane-placental junction or a “l-sign” (which suggests dichorionic twins) [10], whereas fetal sex was identified by checking for the sagittal sign, as described previously [11,12]. All ultrasonographic examinations were performed by wellexperienced ultrasonographic technicians, and all images were reviewed by gynecologists to confirm the diagnosis. The treatment characteristics and outcomes of each IVF cycle were recorded and stored in a computer database. Chi-square tests were used to analyze the effect of each proposed risk factor on the incidence of MZT. A p value <0.05 was considered statistically significant. Results The total number of IVF cycles from January 2001 to December 2011 with a positive pregnancy result was 1338, in which 17 pairs of MZT were identified, resulting in an overall incidence of MZT of 1.3% in this study. All 17 pairs of MZT resulted from the transfer of fresh embryos. Only one pair of MZT resulted from the use of donated oocytes, and the rest of the MZT pairs resulted from embryo transfer of autologous oocytes. We did not analyze these two groups separately because the number of MZT pairs resulting from donated oocytes was small. The summary of patient characteristics, treatment protocols, and chorionicities of the 17 MZT pregnancies is shown in Table 1, and the proposed MZT risk factors with their respective incidences of MZT gestation are summarized in Table 2. The mean maternal age of MZT pregnancies was 32.1 years. To analyze the effects of maternal age on the incidence of MZT pregnancy, we compared IVF cycles performed in women aged <35 years with women 35 years. The results showed that the incidence of MZT pregnancy was higher in cycles performed in women aged <35 years, although the difference was not statistically significant (1.5% vs. 0.8%, respectively; p ¼ 0.319). The effect of ICSI on the incidence of MZT pregnancy was analyzed. There were 853 fresh IVF cycles with positive pregnancy results in which ICSI was performed. Twelve ICSI cycles (1.4%) developed MZT gestations. For fresh IVF cycles without ICSI, the incidence of MZT was 1.0%. However, the difference was not statistically significant (p ¼ 0.620). We also examined the effect of AH on the incidence of MZT pregnancy, but the results showed no increase in MZT incidence in the AH group compared to those without AH (0.9% vs. 2.1%). The influence of embryo culture length on the incidence of MZT pregnancy was also reviewed. The incidence of MZT pregnancy in the Day 5 group was 1.4% (2 out of 151), which is not significantly different from the 1.3% (15 out of 1191) incidence in the cleavage stage group. The chorionicity of MZT pregnancies was determined via transvaginal ultrasonographic examinations performed in the first and/or second trimester (Figure 1). Two MZT gestations had undetermined chorionicities due to early fetal demise. Ten (67%) of the MZT pairs with known chorionicities were monochorionic, whereas five (33%) were dichorionic. Of the monochorionic MZT pregnancies, seven (70%) were diamniotic and three (30%) were monoamniotic. The obstetric outcomes of all 17 cases with MZT pregnancies are summarized in Table 3. Fourteen cases resulted in at least one live birth, including those with coexisting heterozygotic infant (s). In six of these cycles, at least one monozygotic baby was born alive. A coexisting viable heterozygotic infant was delivered at the same time in two cases. Only one pair of monochorionicemonoamniotic
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Table 1 Summary of patient characteristics, treatment protocols, and chorionicities. Case no.
Maternal age (y)
Type of infertility
Etiology
IVF protocol
ICSI
AH
Day of ET
No. of embryos transferred
OHSS
Chorionicity
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
33 28 29 32 32 35 28 31 32 33 31 36 37 33 34 30 41
Secondary Primary Primary Primary Primary Secondary Primary Secondary Secondary Primary Primary Primary Primary Primary Secondary Primary Primary
Tubal factor Male factor Male factor Tubal factor Unexplained Tubal factor Tubal factor Ovulatory factor Ovulatory factor Unexplained Combined Male factor Male factor Male factor Tubal factor Ovulatory factor Ovulatory factor
Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Long agonist Antagonist Antagonist Long agonist Long agonist Antagonist Antagonist Antagonist
þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
3 3 3 3 2 3 2 2 3 2 3 3 3 2 5 4 5
5 6 5 3 2 3 3 3 4 4 3 3 4 2 4 3 3
þ þ
DCDA MCDA MCDA MCDA DCDA DCDA MCMA MCDA MCDA MCMA MCMA Unknown Unknown DCDA DCDA MCDA MCDA
AH ¼ assisted hatching; DCDA ¼ dichorionicediamniotic; ET ¼ embryo transfer; ICSI ¼ intracytoplasmic sperm injection; MCDA ¼ monochorionicediamniotic; MCMA ¼ monochorionicemonoamniotic; OHSS ¼ ovarian hyperstimulation syndrome.
(MCMA) twins survived in this study (Case 11). In particular, one case developed monozygotic triplets, and all three babies were delivered alive (Case 17). In the three cases in which there was no live birth, one had a coexisting singleton and resulted in triplet fetal deaths during the first trimester. The other two cases with only MZT fetuses acquired TTTS; both were monochorionicediamniotic (MCDA) twins and each received an elective termination at 26 weeks and 30 weeks of gestation, respectively. A total of nine cases received an elective fetal reduction, whereas spontaneous fetal loss occurred in six. In summary, a total of 58 fetuses developed from 17 IVF cycles with MZT pregnancies. Thirty-nine were monozygotic and 19 were coexisting singletons or twins. Eleven of 39 (28%) monozygotic babies and 16 of 19 (84%) coexisting heterozygotic fetuses were born alive. Discussion Monozygotic twin pregnancies have an increased risk of obstetric complications and poor pregnancy outcomes. Although it is a rare event, its incidence had been reported in previous studies to increase by as much as four-fold as a result of ART procedures [1,4,5,13,14]. The trend towards late marriage and delaying conception has resulted in the increased practice of ART in many countries worldwide. This phenomenon is even more markedly demonstrated in Taiwan by the increase of total IVF cycles performed from 2001 to 2010 (from 6458 to 11,513, a 78% increase). The percentage of live births resulting from IVF cycles has also increased from 0.8% to 1.8% [15]. Therefore, MZT gestations may become an issue as a growing number of ART practices persist. Unlike dizygotic multiple pregnancies, which can be easily explained by multiple ovulations or multiple embryo implantations, the actual mechanism resulting in the development of Table 2 Summary of proposed monozygotic twinning (MZT) risk factors with their respective MZT incidences. Proposed MZT risk factor
Maternal age > 35 y ICSI Assisted hatching Day-5 transfer
MZT incidence (%)
p
With risk factor
Without risk factor
0.82 1.40 0.94 1.35
1.53 1.02 2.10 1.26
ICSI ¼ intracytoplasmic sperm injection.
0.319 0.620 0.103 1.000
monozygotic multiple pregnancies remains uncertain. It seems obvious that the moment of embryonic cleavage plays an important part in the outcome. However, there is not yet a single theory explaining the mechanism of zygotic splitting that is responsible for the increased incidence of MZT as a result of various assisted reproduction methods. Several aspects of ART techniques have been discussed previously in an attempt to link their associations with MZT incidence, including maternal age, ICSI, AH, PGD, and length of embryo culture. In the current study, the overall incidence of MZT pregnancy was 1.3%, a result comparable to previously reported data, which was approximately three times higher than a rate of 0.4% from natural conceptions [1,16e18]. Previous reports have suggested that advanced maternal age increases the incidence of spontaneous MZT pregnancies [16]. It has been proposed that the average thickness of the ZP gradually decreases with increasing maternal age [19e22], which may alter the normal hatching process, for instance, the splitting of blastocyst during its protrusion due to multiple sites of zona lysis [23]. However, two recent studies reported outcomes that conflict with the concept of advanced maternal age being a risk factor [24,25]. The correlation between zona properties and maternal age was further challenged by two other recent published reports [26,27]. Interestingly, our data also showed a trend towards a higher incidence of MZT pregnancies in women aged <35 years compared to those aged 35 years (1.5% vs. 0.8%; p ¼ 0.319). The use of micromanipulation in ART has been frequently linked with the development of MZT. The proposed mechanism for this link is related to ZP damage that creates gaps, which induce herniation in the blastomere membranes, causing embryo splitting. Previous reports compared MZT incidences between ZP manipulation procedures such as ICSI or AH and conventional IVF. Although some studies found an increased incidence of MZT pregnancy after ZP manipulation [2,23,28e30], many others did not observe this association [14,18,31e34]. In the current study, we also failed to observe significant difference in MZT incidence between IVF with zona manipulation and conventional IVF. The contradictory results between the various studies may be due to the following reasons. First, most of the studies, including the current report, were carried out as retrospective reviews. The significant heterogeneity between these study groups makes it difficult to compare and analyze the results objectively. Furthermore, there were various reported techniques of performing ZP manipulation, and the procedures used in the earlier reports might have
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Fig. 1. Summary of documented chorionicity of monozygotic twin (MZT) pregnancies.
been replaced by newer techniques described in later studies [9]. The question of whether different techniques of micromanipulation procedures can affect the incidence of MZT pregnancy remained to be answered. Finally, there may exist unknown mechanisms during IVF procedures, other than ZP manipulation, that are involved in embryo splitting. The other factor being discussed that may affect the MZT pregnancy incidence is the stage at which the embryo is transferred. With the introduction of sequential culture media, blastocyst transfer has gained increasing popularity over the past decade [35]. It has long been accepted that transferring an embryo at the blastocyst stage enhances implantation rates. In this way, the number of transferred embryos can be reduced to lower the risk of multiple pregnancies without decreasing the pregnancy rate [36]. Nevertheless, many studies have reported an increased risk of MZT pregnancies after a blastocyst transfer compared with a cleavage stage embryo transfer [24,29,32,37e39]. This increase has been explained by several mechanisms, including prolonged culture time [40], culture media composition [41,42], and the experience of laboratory embryologists [37,43]. By contrast, other recent studies did not find an increase in MZT pregnancy rate after blastocyst transfer [44,45]. Our findings also showed no significant difference in MZT risk between the two stages of embryo transfer. Again, many of these studies were small series studies, which resulted in great variations in MZT rates. Further larger trials are required to investigate whether the stage at which the embryo is transferred has an impact on MZT rate.
To evaluate the obstetric outcomes of MZT pregnancies, we discuss not only the effect of IVF procedures on the incidence of MZT pregnancy but also the membrane configuration of each MZT cycle. In the current study, the percentage of each type of chorionicity (dichorionicediamniotic, MCDA, and MCMA) was 33.3%, 46.7%, and 20.0%, respectively. In particular, the incidence of MCMA twins was much higher than those from spontaneous MZT (1e2%) [46]. This finding is similar to the result of a previous study that reported the significantly increased incidence of MCMA twins (37%) after ART treatments [13]. Although the relative occurrence of each type of chorionicity of ART-derived MZT requires further supporting data, the results raise concerns that the obstetric risk may be elevated in women receiving ART procedures. MCMA twins are well known for their high risk of cord entanglement and TTTS. As a consequence, previous reports have emphasized the high perinatal loss rate in MCMA twins (range, 19e44%) [47e50]. Fortunately, the three cases of MCMA twin pregnancies in our study had relatively good outcomes. Two received elective fetal reduction and resulted in live birth deliveries of coexisting heterozygotic twins. One had a coexisting singleton delivered alive with the MCMA twins, which made them the only surviving MCMA twins in this study. By contrast, the only two cases with TTTS had MCDA twins. Both received an elective termination during the early third trimester due to poor fetal conditions. In conclusion, this study represents the first single-center experience that analyzes the relationship between MZT incidence and ART in Taiwan. In addition, the pregnancy outcomes were also
Table 3 Summary of pregnancy outcomes. Case no.
Monozygotic twins/triplets
Coexisting heterozygotic infant (s)
Delivery method
Gestational age (wk)
Birth weight (g)
Sex
1
Fetal reduction 2
Cesarean
34
1540, 2080
F, M
2 3 4 5 6 7 8 9
Expired 2 Expired 2 IUFD 3 Fetal reduction Fetal reduction Fetal reduction Alive 2 Fetal reduction
Fetal reduction 1 Alive 2 0 0 0 0 Alive 2 Alive 2 0 Alive 1
Vaginal Vaginal Vaginal Cesarean Cesarean Cesarean Cesarean Cesarean
30 26 11 31 36 34 32 27
1120/899 695/775 10/5/5 1350/1610 2550, 1865 2020, 1850 2260/2250 1039
F/F Ambiguous Ambiguous M, F F, F M, F M/M F
10 11 12 13 14 15 16 17
Fetal reduction 4 Alive 2 Fetal reduction 2 Alive 1, IUFD 1 Alive 1, IUFD 1 Fetal reduction 2 Fetal reduction 2 Alive 3
Cesarean Cesarean Cesarean Cesarean Cesarean Cesarean Vaginal Cesarean
35 34 40 35 36 36 38 31
2390, 950 1745/1670, 1830 3140 2555, 2375 2750, 2190 2070, 2095 2890 1740/1285/1560
M, M M/M, F M F, F F, M F, M M M/M/M
2, Alive 2 2 2 2
Alive Alive Alive Alive Alive Alive Alive 0
2 1 1 1 1 2 1
IUFD ¼ intrauterine fetal death; TTTS ¼ twinetwin transfusion syndrome.
Complication
At least 1 live birth þ
TTTS TTTS
Maternal pulmonary edema
þ þ þ þ þ þ þ þ þ þ þ þ þ
328
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summarized. Our results concur with previously reported data that the rate of MZT is higher in women receiving ART compared with that of spontaneous MZT. Our study also agreed with recent findings that MZT pregnancies may be associated with younger women. However, we did not find a significant increase in the MZT rate from IVF cycles with micromanipulation or blastocyst transfer. Lastly, the incidence of MCMA twins (which is believed to be the rarest form of MZT) appeared to increase significantly after ART. There are several limitations to this study that must be discussed. Similar to many of the previous studies, the current study was carried out through a retrospective review. In addition, different age groups might receive different ART, which makes objective comparisons difficult. Furthermore, the diagnosis of MZT was based on the findings of the first or second trimester ultrasound, which identified those with a number of fetuses exceeding the number of embryos transferred. The confirmation of MZT through neonatal DNA analysis was not performed in any of the cycles. This fact may lead to the underestimation of MZT, as some MZT might have been misinterpreted as fraternal twins in cases of dichorionicediamniotic twins. Lastly, this study was conducted based on a single center experience, which provided us with a relatively small sample size. The rare occurrence of MZT incidence also resulted in the paucity of cases of interest. Further larger trials are required to draw solid conclusions on this topic. MZT has long been associated with the use of ART. Based on the findings of current study and previous studies, patients receiving IVF should not be overly concerned about the increase in MZT risk when proceeding to various other ART (i.e., ICSI, AH, and blastocyst transfer). However, the incidence of MCMA twins may be significantly increased after IVF/ICSI cycles. We suggest the initiation of national scale multicenter trials to assess the relative risks of each proposed contributing factor of MZT. Through a better understanding of the relationship between ART and MZT, we will be able to reduce the incidence of MZT and thus lower the potential obstetric risks in future IVF cycles. Conflicts of interest The authors have no conflicts of interest relevant to this article. References [1] Derom C, Vlietinck R, Derom R, Van den Berghe H, Thiery M. Increased monozygotic twinning rate after ovulation induction. Lancet 1987;1:1236e8. [2] 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;74:178e9. [3] Bortolus R, Parazzini F, Chatenoud L, Benzi G, Bianchi MM, Marini A. The epidemiology of multiple births. Hum Reprod Update 1999;5:179e87. [4] Edwards RG, Mettler L, Walters DE. Identical twins and in vitro fertilization. J In Vitro Fert Embryo Transf 1986;3:114e7. [5] Wenstrom KD, Syrop CH, Hammitt DG, Van Voorhis BJ. Increased risk of monochorionic twinning associated with assisted reproduction. Fertil Steril 1993;60:510e4. [6] Sebire NJ, Snijders RJ, Hughes K, Sepulveda W, Nicolaides KH. The hidden mortality of monochorionic twin pregnancies. Br J Obstet Gynaecol 1997;104: 1203e7. [7] Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Kirmeyer S, et al. Births: final data for 2005. Natl Vital Stat Rep 2007;56:1e103. [8] Blake DA, Forsberg AS, Johansson BR, Wikland M. Laser zona pellucida thinningdan alternative approach to assisted hatching. Hum Reprod 2001;16: 1959e64. [9] Mantoudis E, Podsiadly BT, Gorgy A, Venkat G, Craft IL. A comparison between quarter, partial and total laser assisted hatching in selected infertility patients. Hum Reprod 2001;16:2182e6. [10] Sepulveda W, Sebire NJ, Hughes K, Odibo A, Nicolaides KH. The lambda sign at 10-14 weeks of gestation as a predictor of chorionicity in twin pregnancies. Ultrasound Obstet Gynecol 1996;7:421e3. [11] Mazza V, Falcinelli C, Paganelli S, Contu G, Mantuano SM, Battafarano SD, et al. Sonographic early fetal gender assignment: a longitudinal study in pregnancies after in vitro fertilization. Ultrasound Obstet Gynecol 2001;17:513e6.
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