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Incidence and Potential Causes affecting Monozygotic Twin Formation Following in vitro Fertilization and Embryo Transfer
Journal of Reproduction & Contraception 2012 Jun; 24(2):93-101
http://www.RandC.cn [email protected]
Incidence and Potential Causes affecting Monozygotic Twin Formation Following in vitro Fertilization and Embryo Transfer Li LI, Xiao-lin LONG, Hong-zi DU, Wen-hong ZHANG, Yu SHI Assisted Reproductive Department, Institute of Obstetrics and Gynecology of the Third Affiliated Hospital of Guangzhou Medical College, Guangzhou 510150, China
Objectives To study the incidence and potential causes of monozygotic twins after in vitro fertilization and embryo transfer (IVF-ET). Methods A retrospective study was performed on women carrying monozygotic twins (MZTs) after conventional IVF-ET treatment at the Third Affiliated Hospital of Guangzhou Medical College in China from January 2003 to May 2009. The incidence and the miscarriage rate for MZTs following IVF-ET were examined in relation to maternal age, duration of infertility, type and dose of hormone treatment, conventional IVF-ET cycles versus intracytoplasmic sperm injection (ICSI) cycles, the use of fresh or frozen-thawed embryos, and day (post-fertilization) of embryo transfer. Results Sixteen MZT pregnancies occurred in 2 161 patients (incidence of 0.74%), of which 5 miscarried (31.25%). No significant difference was found between MZT and non-MZT groups in terms of maternal age, duration of infertility, duration of gonadotropin (Gn) administration, dosage of Gn, number of oocytes retrieved, number of oocytes fertilized, or number of embryos transferred (P>0.05). The incidence of MZT was not statistically different between conventional IVF-ET cycles and ICSI cycles, between fresh embryos transfer cycles and frozen-thawed embryo cycles, or between different transfer days (P>0.05). Conclusion The incidence of MZTs following IVF-ET treatment greatly exceeds that observed following spontaneous conception. Intracytoplasmic sperm injection, frozenthawed procedures, and embryo transfer on different days were not correlated with an increased incidence of MZT pregnancies. Key words: monozygotic twinning; potential cause; IVF-ET
Corresponding author: Xiao-lin LONG; Tel: +86-21-81292233; Fax: +86-21-81292457; E-mail: [email protected] 93
Conference Information 2012 Annual Meeting of International Urogynecological Association September 4th to 8th Australia / Brisbane Obstetrics/Gynecology, Urology Contact: International Urogynecological Association E-mail: [email protected] Website: http://www.iuga.org/?page=2012meeting 21st National Conference on Incontinence & 37th Annual Meeting of IUGA September 4th to 9th Australia / Brisbane Obstetrics/Gynecology, Other Specialties, Urology Contact: Kendra Busby, International Urogynecological Association Phone: +954-763-1456 Fax: +954-763-1236 E-mail: [email protected] Website: http://www.iuga2012.com/ Canadian Fertility & Andrology Society 2012 Annual Meeting September 6th to 9th Ontario / Ottawa Endocrinology, Obstetrics/Gynecology, Urology Contact: Canadian Fertility and Andrology Society Phone: +514-524-9009 Fax: +514-524-2163 E-mail: [email protected] Website: http://www.cfas.ca/index.php?option=com_content&view=article&id=884&Itemid=702 21st Biennial Conference on Diseases of the Vulva and Vagina September 7th to 9th Illinois / Chicago Obstetrics/Gynecology Contact: Debbie Roepe, Executive Director, International Society for the Study of Vulvovaginal Disease Phone: +704-814-9493 Fax: +704-814-9571 E-mail: [email protected] Website: https://netforum.avectra.com/eWeb/StartPage.aspx?Site=ISSVD Basic Genetics for ART Practitioners September 7th Italy / Rome Obstetrics/Gynecology, Other Specialties Contact: Organizing Secretariat, ESHRE Central Office Phone: +11-32-2-269-0969 Fax: +11-32-2-269-5600 E-mail: [email protected] Website: http://www.eshre.eu/ESHRE/English/Calendar/ESHRE-Campus-2012/Basic-genetics-for-ART-practitioners/page.aspx/1278
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In natural pregnancy, monozygotic twinning occurs at an incidence of approximately 0.42%[1]. The incidence of MZT following successful ovarian stimulation cycles and in vitro fertilization-embryo transfer (IVF-ET) cycles is about 0.72%-2.59% which is significantly higher than that in physiological pregnancy[2], though the mechanisms for this increase are still unclear. At present, these studies suggest that this higher incidence of MZT in IVT-ET was related to maternal age[2], microscopic procedures on the zona pellucida of embryos[3,4], the embryo frozen-thawed procedure, or to longer-term culture to the blastula stage before transfer [5-8]. We retrospectively analyzed the incidence and miscarriage rates of MZT pregnancies after conventional IVF, ICSI, or frozen-thawed embryo transfer (FET) in our clinic over the period from January 2003 to May 2009. We examined possible causes contributing to MZT incidence by testing statistical correlation with a number of maternal factors and treatment variables.
Materials & Methods Subjects A total of 2 161 successful IVF/ICSI-ET treatment cycles at the infertility clinic of the Third Affiliated Hospital of Guangzhou Medical College were considered in this study. These cases included 1 277 cycles of conventional IVF, 394 cycles of ICSI, and 490 cycles of FET. Study methods A conventional long agonist protocol during luteal phase was used in IVF-ET. Patients were given 1.3-1.8 mg triptorelin (Ipsen, France) via intramuscular injection in mid-luteal phase. When proper down-regulation was achieved (E2 < 50 pg/ml; endometrial thickness < 5 mm, and maximum follicular diameter < 5 mm), recombinant follical stimulating hormone (rFSH, Gonal-F, Serono, Switzerland) or human menopausal gonadotropin (hMG, Livzon Pharmaceutical Group Inc.) was injected intramuscularly on day 3 of the menstrual cycle to promote follicular development. The dose of Gn was modified based on the development of follicles. When 2 follicles or more were ≥ 18 mm in diameter in both ovaries, Gn was discontinued. In the evening of that day, human chorionic gonadotropin (hCG, 10 000 U, Livzon Pharmaceutical Group Inc.) was injected intramuscularly at 22∶30. Oocytes were obtained 34-36 h later, followed by conventional fertilization or ICSI 3-4 h later. After fertilization, embryos were cultured for 48-72 h using G3 series and G5 series medium (Vitrolife, Switzerland), 1-3 embryos was transferred on day 2, day 3, or day 5 after fertilization. Embryos were cryopreserved by programmed freezing or vitrification, and were transferred on the day of embryo recovery. After transfer, hCG or progesterone (Zhejiang Xianju Pharmaceutical Co., LTD) was injected for luteal support. Urine hCG testing was performed 2 weeks after the transfer and luteal support was continued in those patients with 94
positive results. Transvaginal ultrasound was conducted 30 d after the transfer, and those patients showing a gestational sac were diagnosed with clinical pregnancy. Diagnosis of MZT pregnancy Patients who had a larger number of fetuses than embryos transferred or who had two active fetal hearts within a single gestational sac as indicated by transvaginal ultrasound were diagnosed as carrying MZ twins. Statistical methods The SPSS 13.5 software package was used for statistical analyses. Incidence of MZT during IVF-ET cycles and putative factors including the occurrence of MZT during IVF-ET cycles were analyzed by χ2 test and t test, respectively. P<0.05 was considered to be significantly different.
Results Of the 2 126 treatment cycles that resulted in clinical pregnancy, there were 1 277 cycles of IVF, 394 cycles of ICSI, and 490 cycles of FET. There were 1 508 single pregnancies (69.78%) and 653 multiple pregnancies (30.22%). Of these multiple pregnancy patients, 16 had MZT, accounting for 0.74% of all pregnancies and 2.45% of all multiple pregnancies. Monozygotic twins were seen in 13 patients undergoing IVF of fresh cycles, 2 patients undergoing the ICSI of fresh cycles, and 1 patient undergoing a frozen-thawed embryo transfer. No statistically significant differences (P>0.05) were found between MZT and non-MZT groups in terms of maternal age, duration of infertility, duration of gonadotropin (Gn) administration, dosage of Gn, number of oocytes retrieved, number of oocytes fertilized, or number of embryos transferred (Table 1). Table 1 Comparison of patient history and IVF treatment parameters between MZT and non-MZT mothers (x- ±s) Group
Age (year)
Duration of infertility (a)
MZT
29.1 ± 2.1 Non-MZT 31.0 ± 3.7 P 0.06
Duration of Gn (d)
Dosage of Gn
No. of oocytes
No. of oocytes
No. of embryos
(ampoules)
obtained
fertilized
transferred
4.4 ± 2.6
11.3 ± 1.1
29.6 ± 5.6
16.6 ± 7.6
12.6 ± 6.2
2.1 ± 0.4
4.8 ± 2.9
11.3 ± 1.9
32.3 ± 10.7
14.1 ± 6.8
10.9 ± 6.0
2.3 ± 0.5
0.62
0.98
0.34
0.17
0.28
0.20
Furthermore, no significant difference (P>0.05) in MZT incidence was found between conventional IVF cycles and ICSI cycles, between fresh embryo cycles and frozen-thawed embryo cycles, and between patients who received embryos transferred 2, 3, or 5 d after fertilization (Tables 2-4). 95
Table 2 Incidence of MZT following conventional fresh IVF cycles and fresh ICSI cycles Group MZT
IVF 1% (13/1277)
Non-MZT
ICSI 0.5% (2/394)
99% (1 264/1 277)
99.5% (392/394)
Table 3 Incidence of MZT following embryo transfer between fresh cycles and frozen-thawed cycles Group MZT Non-MZT
Fresh cycles
Frozen-thawed cycles
0.9% (15/1 671)
0.2% (1/490)
99.1% (1 656/1 671)
99.8% (489/490)
Table 4 Incidence of MZT on different post-fertilization transfer days Group MZT Non-MZT
D2 0.2% (1/407) 99.8% (406/407)
D3 1.3% (15/1 121) 98.7% (1 106/1 121)
D5 0% (0/143) 100% (143/143)
All 16 patients who had MZT received more than one embryo to transfer. Of these patients, 3 had twin pregnancy, 11 had triplet pregnancy, and 2 had quadruplet pregnancy. As of October 2009, 2 patients with twin pregnancy had normal delivery, 7 patients with triplet or quadruplet pregnancy had normal delivery with 8 babies after embryo reduction, 2 patients had ongoing pregnancy, and 5 experienced miscarriage (31.25%). Among miscarriage patients, one with quadruplet pregnancy declined embryo reduction and experienced twinto-twin transfusion syndrome and subsequent miscarriage.
Discussion Incidence of MZT in IVF-ET cycles With the widespread use of assisted reproductive technologies, the incidence of multiple pregnancy has increased markedly. Indeed, IVF results in multiple pregnancy 2-6 times more often then physiological pregnancies[9,10]. In this retrospective analysis, we observed an incidence of 0.74% for MZT among 2 161 treatment cycles that resulted in clinical pregnancy, also significantly higher than that observed in physiological pregnancies[1] and within the elevated range reported following IVF. Moreover, these number may be an underestimation because some dichorionic diamniotic MZTs are not distinguishable by ultrasound at early stages following IVF/ICSI-ET. In fact, studies have indicated that MZT incidence could be up to 5% after conventional IVF. The accuracy of MZT diagnosis could be improved by performing single embryo transfer [1]. In China, however, the cost of IVF therapy is relatively expensive and not covered by health insurance. Therefore, patients 96
seeking pregnancy through assisted reproductive technologies are usually not willing to receive single embryo transfer. Mechanisms underlying MZT occurrence following IVF-ET The molecular mechanisms for monozygotic twinning, where two genetically identical embryos are derived from the same oosperm, are still largely unknown. If the oosperm divides within 3 d after fertilization, dichorionic diamniotic (DCDA) MZ twins are formed. In contrast, mono-chorionic diamniotic (MCDA) MZ twins are formed if division occurs 4-8 d after fertilization, while mono-chorionic mono-amniotic (MCMA) MZ twins will form if division occurs on day 8 or later after fertilization. It is currently believed MZT formation may be related to factors that give rise to disruption of the connection within the blastomere or inner cell mass; these factors cause the nascent embryo to divide into two embryos and develop independently[12]. Causes of MZT following IVF-ET The specific causes leading to the higher MZT incidence following IVF-ET are still unclear. Based on previous reports and data from our center, we analyzed the following candidates: maternal age, ovarian stimulating drugs, ICSI and other procedures that affect the zona pellucida, embryo freezing, and embryo culture conditions and duration. Maternal age Recent studies have yielded mixed results on the correlation between MZT occurrence and maternal age. Bulmer[13] found that the incidence of MZT was 12%-22% higher in the women aged 35 years or older compared with the mothers aged 25 years or younger. Abusherkha et al.[2] also demonstrated an increased MZT incidence in the >35-year group following IVF/ICSI, though the difference was statistically insignificant (P>0.05). Alikani et al. [14] reported a tendency towards increased MZT incidence with maternal age. Other epidemiological studies, however, demonstrated that maternal age had no impact on MZT incidence[4,15]. Similarly, we compared MZT pregnancy and non-MZT pregnancy across age groups and there was no significant difference. In addition, all mothers with MZT pregnancy were younger than 35 years, suggesting that MZT incidence following IVF-ET might not be closely related to maternal age. Administration of ovarian stimulating drugs Kallen et al.[16] demonstrated that MZT incidence increased by up to 2 folds following ovulation stimulation cycles compared with natural cycles. Derom et al. [17] compared pregnancy outcome after ovulation stimulation with oral clomiphene with a control group and also found a higher MZT incidence when ovulation stimulation was employed. In addition, the authors suggested that the increased MZT incidence might be related to the delayed fertilization during ovulation stimulation cycles and the changes in the hardness of the zona pellucida caused by ovarian stimulating drugs. 97
Microscopic procedures on zona pellucida Both ICSI and chemical, laser, or mechanically assisted hatching (AH) procedures impair the integrity of the zona pellucida. If an artificial fissure is not properly created, the inner cell mass may be partially trapped in the fissure when the blastula is hatched, dividing the embryo into two parts and leading to MZT. Tarlatzis et al. [18] reported a significantly higher incidence of MZT following ICSI cycles than following conventional IVF cycles. In contrast, Sills et al.[19] reported an insignificant difference in MZT incidence following microscopic procedures on the zona pellucida compared with untreated embryos and instead suggested that transfer of multiple embryos was more likely to result in MZT than subsequent microscopic treatment of the zona pellucida on embryos. It has also been suggested that the MZT incidence following microscopic procedures on the zona pellucida was related to the size and number of fissures created. Frankfurter et al. [20] found an MZT incidence of only 1.2% after transferring blastulae without zone pellucidae and suggested that there were more likely causes of MZT. In the present report, MZT incidence was higher in IVF cycles (1%) than in ICSI cycles, but the difference was not significant, suggesting MZT occurrence might not be related to microscopic procedures on the zona pellucida. Frozen-thawed cycles and other changes in embryo temperature In 1921, Stockard[21] first demonstrated that changes in temperature led to increased MZT pregnancies in live-bearing fish. In IVF, embryos are transferred either immediately or following a frozen-thawed cycle, and some studies reported an increase in MZT incidence following transfer of frozen-thawed embryos[10,22]. This difference in MZT incidence may reflect changes in hardness of the zona pellucida, rather than the direct effect of temperature per se on embryonic development[12]. We found no statistically significant difference in MZT incidence between IVF pregnancies following transfer of fresh or frozen-thawed embryos. In fact, MZT incidence was higher following transfer of fresh embryos, similar to the results reported by Alikani et al.[14] Oocyte retrieval, fertilization, degranulation and observation of embryo morphology, and the repeated removal and return of embryo cultures from the 37℃ incubator might have impacted the hardness of zona pellucida and the development of the embryos, but effects of these more modest temperature changes have not been systematically studied. Culture conditions and duration Chida et al.[23] found that 3.1% of 261 mouse blastulae fertilized in vitro had a double inner cell mass (ICM), whereas a double ICM was observed in only 0.64% of blastulae cultured from 2-cell embryos. They believed that this higher incidence of double ICM might be caused by abnormal fertilization during in vitro culture. Cassuto et al.[24] found that prolonged in vitro culture did not lead to increased MZT incidence, but that the use of ISM1/ ISM2 and ISM1/ISM3 culture medium with prolonged culture to the blastula stage was associated with a 10-fold increase in MZT incidence compared with embryos cultured for 98
2 d or 3 d. These authors suggested that this effect was related to increased apoptosis induced by the higher concentration of glucose and lower sulfide amino acids in ISM3. With short-term culture (to 2 d or 3 d), activated radical-clearing mechanisms in the medium could partially offset these adverse effects, while long-term culture to the blastula stage could disrupt inter-cellular connections in the ICM, leading to cell separation and a higher MZT incidence. In our study, MZT incidences were compared for embryos transferred on days 2, 3, and 5. No significant differences were found among three groups. No MZTs occurred following day 5 embryo transfer. This independence of MZT incidence on transfer day may relate to the relatively minor differences between the G3 and G5 series serum which were used for the culture of embryos, which would have a minimal impact on the structure and function of a developing embryo. In this study, fewer patients were subjected to transfer on day 5, and none showed MZT. Furthermore, the overall incidence of MZT was low, and this rarity is a major obstacle impeding delineation of the mechanisms of MZT. Different methods for early recognition of MZT and their clinical value Monozygotic twins can cause a number of maternal and perinatal complications, so early recognition is particularly important. Due to the presence of placental communicating vessels, mono-chorionic twin pregnancy is associated with higher morbidity and mortality (30%-70%) than dichorionic twin pregnancy. Early recognition and fetal monitoring can reduce morbidity and mortality to 8%[25]. Knowledge of chorion status not only facilitates the monitoring of high risk pregnancies, but also benefits treatment decisions and helps reduce complications. Early recognition of MZT is mainly achieved by B-mode ultrasound. Transvaginal ultrasound offers earlier recognition and more precise results than abdominal ultrasound, including identification of fetal gender, number of fetuses, and the thickness of the inter-twin chorion. At weeks 6-7, transvaginal ultrasound may reveal a substantial sac with a double ring; within the sac is a smooth light band separating the sac into two small chambers, each with germ and primitive cardiac activity. At weeks 9-10, high resolution transvaginal ultrasound between 9 and 10 weeks gestation is a very precise way of determing the type of amniotic sac and chorion. An evidence indicates diamnotic monochorionic twin pregnancy with a single chorion and two amniotic sacs separated by a thin “intertwin” membrane measuring less than 2 mm in thickness. While a thickness >2 mm between the two amniotic sacs indicates dichorionic diamniotic twin pregnancy with absence of the lambda sign. Potential causes and prevention of the higher miscarriage rate in MZT In our study, all 16 patients with MZT received transfer of more than one embryo. Only 2 patients with twin pregnancy had normal delivery. Seven patients with triplet or quadruplet pregnancy had normal pregnancy after embryo reduction and 2 patients had ongoing pregnancy, while 5 had miscarriages (31.25% of all MZT patients). Multiple pregnancy can cause numerous maternal and perinatal complications, including hypertensive disorders, intrahepatic cholestasis, hydramnion, premature rupture of membrane, placental abruption, fetal anomaly, 99
and fetal growth restriction, leading to much higher incidences of miscarriage, preterm, and perinatal death compared with single pregnancy. Besides the usual complications of multiple pregnancy, MZT is also associated with unsynchronized development between the 2 fetuses (twin-to-twin transfusion syndrome) and abnormal fetal development, which further increase the risk for poor pregnancy outcome. In IVF-ET, transfer of multiple embryos may be one of the causes for increased incidence of multiple pregnancy and MZT[9]. According to regulations imposed by the Chinese Ministry of Health, 2 embryos can be transferred in the first attempt in patients younger than 35 years, while 3 can be transferred to older patients or in the second attempt. Therefore, the risk of MZT occurrence can be reduced by decreasing the number of embryos transferred. Some authors have speculated that[26,37], while it was safe to transfer 2 embryos per IVF-ET cycle, single-blastula transfer might be more efficient. For multiple pregnancy accompanied by MZT, embryo reduction during early pregnancy is another option. In principle, the MZT gestational sac should be preferentially eradicated; for patients with MZT alone, prenatal monitoring and diagnosis should be reinforced. Another option is to interrupt placental communicating vessels between the twins using electrocoagulation to reduce twin-to-twin transfusion syndrome. In summary, MZT incidence was higher following IVF-ET treatment than observed during natural pregnancy. The microscopic ICSI procedure, frozen-thawed procedure, and day of embryo transfer were not related to increased MZT incidence. Overall MZT incidence is low in both natural cycles and IVF cycles, however, it is difficult to investigate the causes and mechanisms of MZT occurrence. Therefore, these results must be confirmed by multi-center, longer-term studies involving a larger sample size.
References 1. Rao A, Sairam S, Shehata H, et al. Obstetric complications of twin pregnancies. Best Pract Res Clin Obstet Gynaecol, 2004, 18(4):557-76. 2. Abusheikha N, Salha O, Sharma V, et al. Monozygotic twinning and IVF/ICSI treatment: a report of 11 cases and review of the literature. Hum Reprod Update, 2000, 6(4):396-403. 3. Schieve LA, Meikle SF, Peterson HB, et al. Does assisted hatching pose a risk formonozygotic twinning in pregnancies conceived through in vitro fertilization? Fertil Steril, 2000, 74(2):288-94. 4. Skiadas CC, Missmer SA, Benson CB, et al. 2008 Risk factors associated with pregnancies containing a monochorionic pair following assisted reproductive technologies. Hum Reprod, 2008, 23(6):1366-71. 5. Jain JK, Boostanfar R, Slater CC, et al. Monozygotic twins and triplets in association with blastocyst transfer. J Assist Reprod Genet, 2004, 21(4):103-7. 6. Moayeri SE, Behr B, Lathi RB, et al. Risk of monozygotic twinning with blastocyst transfer decreases over time: an 8-year experience. Fertil Steril, 2007, 87(5):1028-32. 7. da Costa AA, Abdelmassih S, de Oliveira FG, et al. Monozygotic twins and transfer at the blastocyst stage after ICSI. Hum Reprod, 2001, 16(2):333-6. 100
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Milki AA, Jun SH, Hinckley MD, et al. Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer. Fertil Steril, 2003, 79(3):503-6.
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Carrillo-Vadillo R, Garcia-Lozano JC, Lozano-Arana MD, et al. Two sets of monozygotic twins after intracytoplasmic sperm injection and transfer of two embryos on day 2. Fertil Steril, 2007, 88(6):1676e3-e5.
10. Li Z, Zhu GJ, Liu YQ, et al. Monozygotic twinning in IVF-ETs. Journal of Huazhong University of Science and Technology (Health Science)(in Chinese), 2007, 36(5):685-87. 11. Blickstein I, Verhoeven HC, Keith LG. Zygotic splitting after assisted reproduction. N Engl J Med, 1999, 340 (9):738-39. 12. Aston KI, Peterson CM, Carrell DT. Monozygotic twinning associated with assisted reproductive technologies: a review. Reproduction, 2008, 136(4):377-86. 13. Bulmer MG. The Biology of Twinning in Man. Oxford, UK: Clarendon Press, 1970:426-27. 14. Alikani M, Cekleniak NA, Walters E, et al. Monozygotic twinning following assisted conception: an analysis of 81 consecutive cases. Hum Reprod, 2003, 18(9):1937-43. 15. Bortolus R, Parazzini F, Chatenoud L, et al. The epidemiology of multiple births. Hum Reprod Update, 1999, 5(2):179-87. 16. Kallen B, Olausson PO, Nygren KG. Neonatal outcome in pregnancies from ovarian stimulation. Obstet Gynecol, 2002, 100(3):414-9. 17. Derom C, Leroy F, Vlietinck R, et al. High frequency of iatrogenic monozygotic twins with administration of clomiphene citrate and a change in chorionicity. Fertil Steril, 2006, 85(3):755-7. 18. Tarlatzis BC, Qublan HS, Sanopoulou T, et al. Increase in the monozygotic twinning rate after intracytoplasmic sperm injection and blastocyst stage embryo transfer. Fertil Steril, 2002, 77(1):196-8. 19. Sills ES, Moomjy M, Zaninovic N, et al. Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF. Hum Reprod, 2000, l5(4):890-5. 20. Frankfurter D, Hackett R, Meng L, et al. 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(3):144S. 21. Stockard CR. Developmental rate and structural expression: an experimental study of twins,double monsters and single deformities, and the interaction among embryonic organs during their origin and development. Am J Anat, 1921, 28(2):115-277. 22. Faraj R, Evbuomwan I, Sturgiss S, et al. Monozygotic triplet pregnancy following egg donation and transfer of single frozen-thawed embryo. Fertil Steril, 2008, 89(5):1260.e9-e12. 23. Chida S. Monozygous double inner cell masses in mouse blastocysts foll owing fertilization in vitro and in vivo. J In Vitro Fert Embryo Transf, 1990, 7(3):177-9. 24. 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(2):462-3. 25. Monteagudo A, Roman AS. Ultrasound in multiple gestational: twins and other multifefal pregnancies. Clin Perinatal, 2005, 32(2):329-54. 26. Unger S, HoopmannM, Bald R, et al. Monozygotic triplets and monozygotic twins after ICSI and transfer of two blastocysts: case report. Hum Reprod, 2004, 19(1):110-3. 27. Peramo B, Ricciarelli E, Cuadros-Fernández J, et al. Blastocyst transfer and monozygotic twinning. Fertil Steril, 1999, 72(6):1116-7. (Received on April 24, 2012)
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http://www.RandC.cn [email protected]
Incidence and Potential Causes affecting Monozygotic Twin Formation Following in vitro Fertilization and Embryo Transfer Li LI, Xiao-lin LONG, Hong-zi DU, Wen-hong ZHANG, Yu SHI Assisted Reproductive Department, Institute of Obstetrics and Gynecology of the Third Affiliated Hospital of Guangzhou Medical College, Guangzhou 510150, China
Objectives To study the incidence and potential causes of monozygotic twins after in vitro fertilization and embryo transfer (IVF-ET). Methods A retrospective study was performed on women carrying monozygotic twins (MZTs) after conventional IVF-ET treatment at the Third Affiliated Hospital of Guangzhou Medical College in China from January 2003 to May 2009. The incidence and the miscarriage rate for MZTs following IVF-ET were examined in relation to maternal age, duration of infertility, type and dose of hormone treatment, conventional IVF-ET cycles versus intracytoplasmic sperm injection (ICSI) cycles, the use of fresh or frozen-thawed embryos, and day (post-fertilization) of embryo transfer. Results Sixteen MZT pregnancies occurred in 2 161 patients (incidence of 0.74%), of which 5 miscarried (31.25%). No significant difference was found between MZT and non-MZT groups in terms of maternal age, duration of infertility, duration of gonadotropin (Gn) administration, dosage of Gn, number of oocytes retrieved, number of oocytes fertilized, or number of embryos transferred (P>0.05). The incidence of MZT was not statistically different between conventional IVF-ET cycles and ICSI cycles, between fresh embryos transfer cycles and frozen-thawed embryo cycles, or between different transfer days (P>0.05). Conclusion The incidence of MZTs following IVF-ET treatment greatly exceeds that observed following spontaneous conception. Intracytoplasmic sperm injection, frozenthawed procedures, and embryo transfer on different days were not correlated with an increased incidence of MZT pregnancies. Key words: monozygotic twinning; potential cause; IVF-ET
Corresponding author: Xiao-lin LONG; Tel: +86-21-81292233; Fax: +86-21-81292457; E-mail: [email protected] 93
Conference Information 2012 Annual Meeting of International Urogynecological Association September 4th to 8th Australia / Brisbane Obstetrics/Gynecology, Urology Contact: International Urogynecological Association E-mail: [email protected] Website: http://www.iuga.org/?page=2012meeting 21st National Conference on Incontinence & 37th Annual Meeting of IUGA September 4th to 9th Australia / Brisbane Obstetrics/Gynecology, Other Specialties, Urology Contact: Kendra Busby, International Urogynecological Association Phone: +954-763-1456 Fax: +954-763-1236 E-mail: [email protected] Website: http://www.iuga2012.com/ Canadian Fertility & Andrology Society 2012 Annual Meeting September 6th to 9th Ontario / Ottawa Endocrinology, Obstetrics/Gynecology, Urology Contact: Canadian Fertility and Andrology Society Phone: +514-524-9009 Fax: +514-524-2163 E-mail: [email protected] Website: http://www.cfas.ca/index.php?option=com_content&view=article&id=884&Itemid=702 21st Biennial Conference on Diseases of the Vulva and Vagina September 7th to 9th Illinois / Chicago Obstetrics/Gynecology Contact: Debbie Roepe, Executive Director, International Society for the Study of Vulvovaginal Disease Phone: +704-814-9493 Fax: +704-814-9571 E-mail: [email protected] Website: https://netforum.avectra.com/eWeb/StartPage.aspx?Site=ISSVD Basic Genetics for ART Practitioners September 7th Italy / Rome Obstetrics/Gynecology, Other Specialties Contact: Organizing Secretariat, ESHRE Central Office Phone: +11-32-2-269-0969 Fax: +11-32-2-269-5600 E-mail: [email protected] Website: http://www.eshre.eu/ESHRE/English/Calendar/ESHRE-Campus-2012/Basic-genetics-for-ART-practitioners/page.aspx/1278
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In natural pregnancy, monozygotic twinning occurs at an incidence of approximately 0.42%[1]. The incidence of MZT following successful ovarian stimulation cycles and in vitro fertilization-embryo transfer (IVF-ET) cycles is about 0.72%-2.59% which is significantly higher than that in physiological pregnancy[2], though the mechanisms for this increase are still unclear. At present, these studies suggest that this higher incidence of MZT in IVT-ET was related to maternal age[2], microscopic procedures on the zona pellucida of embryos[3,4], the embryo frozen-thawed procedure, or to longer-term culture to the blastula stage before transfer [5-8]. We retrospectively analyzed the incidence and miscarriage rates of MZT pregnancies after conventional IVF, ICSI, or frozen-thawed embryo transfer (FET) in our clinic over the period from January 2003 to May 2009. We examined possible causes contributing to MZT incidence by testing statistical correlation with a number of maternal factors and treatment variables.
Materials & Methods Subjects A total of 2 161 successful IVF/ICSI-ET treatment cycles at the infertility clinic of the Third Affiliated Hospital of Guangzhou Medical College were considered in this study. These cases included 1 277 cycles of conventional IVF, 394 cycles of ICSI, and 490 cycles of FET. Study methods A conventional long agonist protocol during luteal phase was used in IVF-ET. Patients were given 1.3-1.8 mg triptorelin (Ipsen, France) via intramuscular injection in mid-luteal phase. When proper down-regulation was achieved (E2 < 50 pg/ml; endometrial thickness < 5 mm, and maximum follicular diameter < 5 mm), recombinant follical stimulating hormone (rFSH, Gonal-F, Serono, Switzerland) or human menopausal gonadotropin (hMG, Livzon Pharmaceutical Group Inc.) was injected intramuscularly on day 3 of the menstrual cycle to promote follicular development. The dose of Gn was modified based on the development of follicles. When 2 follicles or more were ≥ 18 mm in diameter in both ovaries, Gn was discontinued. In the evening of that day, human chorionic gonadotropin (hCG, 10 000 U, Livzon Pharmaceutical Group Inc.) was injected intramuscularly at 22∶30. Oocytes were obtained 34-36 h later, followed by conventional fertilization or ICSI 3-4 h later. After fertilization, embryos were cultured for 48-72 h using G3 series and G5 series medium (Vitrolife, Switzerland), 1-3 embryos was transferred on day 2, day 3, or day 5 after fertilization. Embryos were cryopreserved by programmed freezing or vitrification, and were transferred on the day of embryo recovery. After transfer, hCG or progesterone (Zhejiang Xianju Pharmaceutical Co., LTD) was injected for luteal support. Urine hCG testing was performed 2 weeks after the transfer and luteal support was continued in those patients with 94
positive results. Transvaginal ultrasound was conducted 30 d after the transfer, and those patients showing a gestational sac were diagnosed with clinical pregnancy. Diagnosis of MZT pregnancy Patients who had a larger number of fetuses than embryos transferred or who had two active fetal hearts within a single gestational sac as indicated by transvaginal ultrasound were diagnosed as carrying MZ twins. Statistical methods The SPSS 13.5 software package was used for statistical analyses. Incidence of MZT during IVF-ET cycles and putative factors including the occurrence of MZT during IVF-ET cycles were analyzed by χ2 test and t test, respectively. P<0.05 was considered to be significantly different.
Results Of the 2 126 treatment cycles that resulted in clinical pregnancy, there were 1 277 cycles of IVF, 394 cycles of ICSI, and 490 cycles of FET. There were 1 508 single pregnancies (69.78%) and 653 multiple pregnancies (30.22%). Of these multiple pregnancy patients, 16 had MZT, accounting for 0.74% of all pregnancies and 2.45% of all multiple pregnancies. Monozygotic twins were seen in 13 patients undergoing IVF of fresh cycles, 2 patients undergoing the ICSI of fresh cycles, and 1 patient undergoing a frozen-thawed embryo transfer. No statistically significant differences (P>0.05) were found between MZT and non-MZT groups in terms of maternal age, duration of infertility, duration of gonadotropin (Gn) administration, dosage of Gn, number of oocytes retrieved, number of oocytes fertilized, or number of embryos transferred (Table 1). Table 1 Comparison of patient history and IVF treatment parameters between MZT and non-MZT mothers (x- ±s) Group
Age (year)
Duration of infertility (a)
MZT
29.1 ± 2.1 Non-MZT 31.0 ± 3.7 P 0.06
Duration of Gn (d)
Dosage of Gn
No. of oocytes
No. of oocytes
No. of embryos
(ampoules)
obtained
fertilized
transferred
4.4 ± 2.6
11.3 ± 1.1
29.6 ± 5.6
16.6 ± 7.6
12.6 ± 6.2
2.1 ± 0.4
4.8 ± 2.9
11.3 ± 1.9
32.3 ± 10.7
14.1 ± 6.8
10.9 ± 6.0
2.3 ± 0.5
0.62
0.98
0.34
0.17
0.28
0.20
Furthermore, no significant difference (P>0.05) in MZT incidence was found between conventional IVF cycles and ICSI cycles, between fresh embryo cycles and frozen-thawed embryo cycles, and between patients who received embryos transferred 2, 3, or 5 d after fertilization (Tables 2-4). 95
Table 2 Incidence of MZT following conventional fresh IVF cycles and fresh ICSI cycles Group MZT
IVF 1% (13/1277)
Non-MZT
ICSI 0.5% (2/394)
99% (1 264/1 277)
99.5% (392/394)
Table 3 Incidence of MZT following embryo transfer between fresh cycles and frozen-thawed cycles Group MZT Non-MZT
Fresh cycles
Frozen-thawed cycles
0.9% (15/1 671)
0.2% (1/490)
99.1% (1 656/1 671)
99.8% (489/490)
Table 4 Incidence of MZT on different post-fertilization transfer days Group MZT Non-MZT
D2 0.2% (1/407) 99.8% (406/407)
D3 1.3% (15/1 121) 98.7% (1 106/1 121)
D5 0% (0/143) 100% (143/143)
All 16 patients who had MZT received more than one embryo to transfer. Of these patients, 3 had twin pregnancy, 11 had triplet pregnancy, and 2 had quadruplet pregnancy. As of October 2009, 2 patients with twin pregnancy had normal delivery, 7 patients with triplet or quadruplet pregnancy had normal delivery with 8 babies after embryo reduction, 2 patients had ongoing pregnancy, and 5 experienced miscarriage (31.25%). Among miscarriage patients, one with quadruplet pregnancy declined embryo reduction and experienced twinto-twin transfusion syndrome and subsequent miscarriage.
Discussion Incidence of MZT in IVF-ET cycles With the widespread use of assisted reproductive technologies, the incidence of multiple pregnancy has increased markedly. Indeed, IVF results in multiple pregnancy 2-6 times more often then physiological pregnancies[9,10]. In this retrospective analysis, we observed an incidence of 0.74% for MZT among 2 161 treatment cycles that resulted in clinical pregnancy, also significantly higher than that observed in physiological pregnancies[1] and within the elevated range reported following IVF. Moreover, these number may be an underestimation because some dichorionic diamniotic MZTs are not distinguishable by ultrasound at early stages following IVF/ICSI-ET. In fact, studies have indicated that MZT incidence could be up to 5% after conventional IVF. The accuracy of MZT diagnosis could be improved by performing single embryo transfer [1]. In China, however, the cost of IVF therapy is relatively expensive and not covered by health insurance. Therefore, patients 96
seeking pregnancy through assisted reproductive technologies are usually not willing to receive single embryo transfer. Mechanisms underlying MZT occurrence following IVF-ET The molecular mechanisms for monozygotic twinning, where two genetically identical embryos are derived from the same oosperm, are still largely unknown. If the oosperm divides within 3 d after fertilization, dichorionic diamniotic (DCDA) MZ twins are formed. In contrast, mono-chorionic diamniotic (MCDA) MZ twins are formed if division occurs 4-8 d after fertilization, while mono-chorionic mono-amniotic (MCMA) MZ twins will form if division occurs on day 8 or later after fertilization. It is currently believed MZT formation may be related to factors that give rise to disruption of the connection within the blastomere or inner cell mass; these factors cause the nascent embryo to divide into two embryos and develop independently[12]. Causes of MZT following IVF-ET The specific causes leading to the higher MZT incidence following IVF-ET are still unclear. Based on previous reports and data from our center, we analyzed the following candidates: maternal age, ovarian stimulating drugs, ICSI and other procedures that affect the zona pellucida, embryo freezing, and embryo culture conditions and duration. Maternal age Recent studies have yielded mixed results on the correlation between MZT occurrence and maternal age. Bulmer[13] found that the incidence of MZT was 12%-22% higher in the women aged 35 years or older compared with the mothers aged 25 years or younger. Abusherkha et al.[2] also demonstrated an increased MZT incidence in the >35-year group following IVF/ICSI, though the difference was statistically insignificant (P>0.05). Alikani et al. [14] reported a tendency towards increased MZT incidence with maternal age. Other epidemiological studies, however, demonstrated that maternal age had no impact on MZT incidence[4,15]. Similarly, we compared MZT pregnancy and non-MZT pregnancy across age groups and there was no significant difference. In addition, all mothers with MZT pregnancy were younger than 35 years, suggesting that MZT incidence following IVF-ET might not be closely related to maternal age. Administration of ovarian stimulating drugs Kallen et al.[16] demonstrated that MZT incidence increased by up to 2 folds following ovulation stimulation cycles compared with natural cycles. Derom et al. [17] compared pregnancy outcome after ovulation stimulation with oral clomiphene with a control group and also found a higher MZT incidence when ovulation stimulation was employed. In addition, the authors suggested that the increased MZT incidence might be related to the delayed fertilization during ovulation stimulation cycles and the changes in the hardness of the zona pellucida caused by ovarian stimulating drugs. 97
Microscopic procedures on zona pellucida Both ICSI and chemical, laser, or mechanically assisted hatching (AH) procedures impair the integrity of the zona pellucida. If an artificial fissure is not properly created, the inner cell mass may be partially trapped in the fissure when the blastula is hatched, dividing the embryo into two parts and leading to MZT. Tarlatzis et al. [18] reported a significantly higher incidence of MZT following ICSI cycles than following conventional IVF cycles. In contrast, Sills et al.[19] reported an insignificant difference in MZT incidence following microscopic procedures on the zona pellucida compared with untreated embryos and instead suggested that transfer of multiple embryos was more likely to result in MZT than subsequent microscopic treatment of the zona pellucida on embryos. It has also been suggested that the MZT incidence following microscopic procedures on the zona pellucida was related to the size and number of fissures created. Frankfurter et al. [20] found an MZT incidence of only 1.2% after transferring blastulae without zone pellucidae and suggested that there were more likely causes of MZT. In the present report, MZT incidence was higher in IVF cycles (1%) than in ICSI cycles, but the difference was not significant, suggesting MZT occurrence might not be related to microscopic procedures on the zona pellucida. Frozen-thawed cycles and other changes in embryo temperature In 1921, Stockard[21] first demonstrated that changes in temperature led to increased MZT pregnancies in live-bearing fish. In IVF, embryos are transferred either immediately or following a frozen-thawed cycle, and some studies reported an increase in MZT incidence following transfer of frozen-thawed embryos[10,22]. This difference in MZT incidence may reflect changes in hardness of the zona pellucida, rather than the direct effect of temperature per se on embryonic development[12]. We found no statistically significant difference in MZT incidence between IVF pregnancies following transfer of fresh or frozen-thawed embryos. In fact, MZT incidence was higher following transfer of fresh embryos, similar to the results reported by Alikani et al.[14] Oocyte retrieval, fertilization, degranulation and observation of embryo morphology, and the repeated removal and return of embryo cultures from the 37℃ incubator might have impacted the hardness of zona pellucida and the development of the embryos, but effects of these more modest temperature changes have not been systematically studied. Culture conditions and duration Chida et al.[23] found that 3.1% of 261 mouse blastulae fertilized in vitro had a double inner cell mass (ICM), whereas a double ICM was observed in only 0.64% of blastulae cultured from 2-cell embryos. They believed that this higher incidence of double ICM might be caused by abnormal fertilization during in vitro culture. Cassuto et al.[24] found that prolonged in vitro culture did not lead to increased MZT incidence, but that the use of ISM1/ ISM2 and ISM1/ISM3 culture medium with prolonged culture to the blastula stage was associated with a 10-fold increase in MZT incidence compared with embryos cultured for 98
2 d or 3 d. These authors suggested that this effect was related to increased apoptosis induced by the higher concentration of glucose and lower sulfide amino acids in ISM3. With short-term culture (to 2 d or 3 d), activated radical-clearing mechanisms in the medium could partially offset these adverse effects, while long-term culture to the blastula stage could disrupt inter-cellular connections in the ICM, leading to cell separation and a higher MZT incidence. In our study, MZT incidences were compared for embryos transferred on days 2, 3, and 5. No significant differences were found among three groups. No MZTs occurred following day 5 embryo transfer. This independence of MZT incidence on transfer day may relate to the relatively minor differences between the G3 and G5 series serum which were used for the culture of embryos, which would have a minimal impact on the structure and function of a developing embryo. In this study, fewer patients were subjected to transfer on day 5, and none showed MZT. Furthermore, the overall incidence of MZT was low, and this rarity is a major obstacle impeding delineation of the mechanisms of MZT. Different methods for early recognition of MZT and their clinical value Monozygotic twins can cause a number of maternal and perinatal complications, so early recognition is particularly important. Due to the presence of placental communicating vessels, mono-chorionic twin pregnancy is associated with higher morbidity and mortality (30%-70%) than dichorionic twin pregnancy. Early recognition and fetal monitoring can reduce morbidity and mortality to 8%[25]. Knowledge of chorion status not only facilitates the monitoring of high risk pregnancies, but also benefits treatment decisions and helps reduce complications. Early recognition of MZT is mainly achieved by B-mode ultrasound. Transvaginal ultrasound offers earlier recognition and more precise results than abdominal ultrasound, including identification of fetal gender, number of fetuses, and the thickness of the inter-twin chorion. At weeks 6-7, transvaginal ultrasound may reveal a substantial sac with a double ring; within the sac is a smooth light band separating the sac into two small chambers, each with germ and primitive cardiac activity. At weeks 9-10, high resolution transvaginal ultrasound between 9 and 10 weeks gestation is a very precise way of determing the type of amniotic sac and chorion. An evidence indicates diamnotic monochorionic twin pregnancy with a single chorion and two amniotic sacs separated by a thin “intertwin” membrane measuring less than 2 mm in thickness. While a thickness >2 mm between the two amniotic sacs indicates dichorionic diamniotic twin pregnancy with absence of the lambda sign. Potential causes and prevention of the higher miscarriage rate in MZT In our study, all 16 patients with MZT received transfer of more than one embryo. Only 2 patients with twin pregnancy had normal delivery. Seven patients with triplet or quadruplet pregnancy had normal pregnancy after embryo reduction and 2 patients had ongoing pregnancy, while 5 had miscarriages (31.25% of all MZT patients). Multiple pregnancy can cause numerous maternal and perinatal complications, including hypertensive disorders, intrahepatic cholestasis, hydramnion, premature rupture of membrane, placental abruption, fetal anomaly, 99
and fetal growth restriction, leading to much higher incidences of miscarriage, preterm, and perinatal death compared with single pregnancy. Besides the usual complications of multiple pregnancy, MZT is also associated with unsynchronized development between the 2 fetuses (twin-to-twin transfusion syndrome) and abnormal fetal development, which further increase the risk for poor pregnancy outcome. In IVF-ET, transfer of multiple embryos may be one of the causes for increased incidence of multiple pregnancy and MZT[9]. According to regulations imposed by the Chinese Ministry of Health, 2 embryos can be transferred in the first attempt in patients younger than 35 years, while 3 can be transferred to older patients or in the second attempt. Therefore, the risk of MZT occurrence can be reduced by decreasing the number of embryos transferred. Some authors have speculated that[26,37], while it was safe to transfer 2 embryos per IVF-ET cycle, single-blastula transfer might be more efficient. For multiple pregnancy accompanied by MZT, embryo reduction during early pregnancy is another option. In principle, the MZT gestational sac should be preferentially eradicated; for patients with MZT alone, prenatal monitoring and diagnosis should be reinforced. Another option is to interrupt placental communicating vessels between the twins using electrocoagulation to reduce twin-to-twin transfusion syndrome. In summary, MZT incidence was higher following IVF-ET treatment than observed during natural pregnancy. The microscopic ICSI procedure, frozen-thawed procedure, and day of embryo transfer were not related to increased MZT incidence. Overall MZT incidence is low in both natural cycles and IVF cycles, however, it is difficult to investigate the causes and mechanisms of MZT occurrence. Therefore, these results must be confirmed by multi-center, longer-term studies involving a larger sample size.
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