- Email: [email protected]
Monozygotic twins in infertile patients with advanced maternal age: case reports and review of the literature
CASE REPORT Monozygotic twins in infertile patients with advanced maternal age: case reports and review of the literature Navid Esfandiari, D.V.M., Ph.D., H.C.L.D.,a,b Monica Kapoor, M.D.,a Hasan Burjaq, B.Sc., M.T.,a Paul Chang, M.D.,a,b Lynda Gotlieb, R.N.,a and Robert F. Casper, M.D.a,b a
Toronto Centre for Advanced Reproductive Technology, and b Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada
Objective: To report two cases of successful monozygotic twin pregnancies in women undergoing infertility treatment and to review possible etiologic factors. Design: Case report and review of the literature. Setting: University of Toronto–affiliated infertility clinic. Patient(s): A 43-year-old woman and a 44-year-old woman with history of secondary infertility. Intervention(s): In vitro fertilization and ET. Main Outcome Measure(s): Monozygotic twin pregnancy. Result(s): Delivery of two sets of monozygotic twins. Conclusion(s): The only identified potential risk factors for monozygotic twins are maternal age and assisted reproductive technologies. Both patients reported here were aged >40 years and underwent assisted reproduction procedures, including high-dose gonadotropin stimulation, embryo culture, and, in case 2, intracytoplasmic sperm injection plus assisted hatching. (Fertil Steril 2009;92:1168.e9–e12. 2009 by American Society for Reproductive Medicine.) Key Words: Monozygotic twinning, maternal age, IVF
Today it is common for women in developed nations to delay childbearing until the fourth decade of life. As fertility declines with age, more couples turn to assisted reproductive technologies (ART) to conceive. Since the first IVF baby born in 1978, this procedure has granted many infertile couples an opportunity for pregnancy, often resulting in an increased incidence of multiple pregnancies (1). At present approximately one third of IVF babies are twins, mostly because more than one embryo is transferred to the uterus to maximize the chance of a successful pregnancy. Dizygotic twins are a known risk of multiple ovulations and multiple embryo transfer. However, data on the etiology of monozygotic twinning (MZT) is limited. Monozygotic twinning is a relatively rare event and is estimated to occur in 0.4% of in-vivo conception (2), compared with approximately 1.5% of all clinical pregnancies after ovulation induction with gonadotropins (3). Monozygotic twinning is Received March 10, 2009; revised May 13, 2009; accepted May 27, 2009; published online July 15, 2009. N.E. has nothing to disclose. M.K. has nothing to disclose. H.B. has nothing to disclose. P.C. has nothing to disclose. L.G. has nothing to disclose. R.F.C. has nothing to disclose. Reprint requests: Navid Esfandiari, D.V.M., Ph.D., H.C.L.D., Toronto Centre for advanced Reproductive Technology, 210-150 Bloor Street West, Toronto, ON M5S 2X9, Canada (FAX: 416-972 0036; E-mail: navid. [email protected]).
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defined according to the stage of development when division of the fertilized ovum occurs. Biamniotic, dichorionic twins occur when division of the embryonic cell mass occurs earlier than 72 hours after fertilization. If division occurs between days 4 to 8 after the inner mass has formed, biamniotic, monochorionic twins will result. Division after day 8 will lead to a monoamniotic, monochorionic twin pregnancy (4). The process of monozygotic twinning is speculated to begin with the protrusion of some trophectoderm cells through a small opening in the zona pellucida (ZP). Some of the cell mass may then break off in the uterus to form a twin pregnancy. Several factors associated with MZT include family history (5, 6), ovarian stimulation (4), the use of intracytoplasmic sperm injection (ICSI), in vitro embryo culture conditions (3, 7), ZP manipulation (7), stage of embryo development at the time of transfer, and maternal age. The first monozygotic pregnancy resulting from IVF was reported by Yovich et al. in 1984 (8). This case study described the outcome of monochorionic, diamniotic twin males. Since the report of this case, there have been many additions to the literature describing similar cases. However, factors that affect the frequency of MZT have been poorly characterized. Monozygotic twinning is considered to be a higher-risk pregnancy that requires additional surveillance compared with dizygotic twinning. The ability to identify risk factors is
Fertility and Sterility Vol. 92, No. 3, September 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2009.05.085
very important to provide adequate counseling and care to the patient. The present case reports describe MZT in two infertility patients with advanced maternal age. CASE REPORTS Case 1 A 43-year-old gravida 1, para 0 patient was referred to our center after 1 year of secondary infertility. She had no prior tubal risk factor and normal results on hysterosalpingogram before her ectopic pregnancy. For her fourth IVF cycle in October 2005 she received a modified microdose flare protocol using letrozole (2.5 mg; Novartis, Dorval, QC, Canada) from day 3 to day 7 of her menstrual cycle, buserelin acetate (Suprefact 0.05 mL SC twice daily; Aventis Pharma, Laval, QC, Canada) starting on day 3, and recombinant FSH (Puregon 300 IU daily; Organon, Scarborough, ON, Canada) starting on day 3. On day 15 of her cycle, 10,000 IU of hCG (Organon) was given. Thirty-six hours later, nine oocytes were retrieved by ultrasound-guided needle aspiration. All oocytes were inseminated 3 hours after the retrieval. Semen analyses revealed a volume of 0.6 mL with a concentration of 34 106/mL and 60% forward progressive motility. The morphology according to World Health Organization (1999) criteria was 18%, with moderate sperm agglutination. The sperm parameters after processing with density gradient was a volume of 0.4 mL with a concentration of 16 106/mL, 98% motility, and 28% normal morphology. Eighteen hours after insemination, only four oocytes were normally fertilized and one oocyte fertilized with 1 pronucleus (PN). The fertilized oocytes were placed in microdrops of P1 (Irvine Scientific, Santa Ana, CA) with 10% synthetic serum substitute under mineral oil (Sigma Chemical, St. Louis, MO) and left in culture for an additional 2 days. All fertilized oocytes cleaved and resulted in one six-cell grade 1 embryo, one four-cell grade 1, one three-cell grade 2, one two-cell grade 2, and one five-cell grade 2 from 1PN fertilization. The grading system used was based on a scale of 1–4, with grade 1 representing highest quality. These five IVF embryos were transferred using a Tomcat catheter (Meditech, Montreal, QC, Canada). Two weeks after ET the patient had a serum b-hCG level of 391 IU/L and 933 IU/L 2 days later. At 8 weeks’ gestation, an ultrasound examination revealed an appropriately sized monochorionic, monoamniotic intrauterine twin pregnancy (Fig. 1) with two fetal heartbeats. The patient delivered twin girls by cesarian section at 34 weeks’ gestation, weighing 2,100 g and 2,300 g, respectively. Hemangioma was observed on the face of one newborn. Case 2 A 44-year-old gravida 1, para 1 patient was referred to our center with 3 years of unexplained secondary infertility. She had mild endometriosis confirmed by laparoscopy. After three failed IVF/ICSI cycles, she began her fourth attempt in April 2006. She received a microdose flare protocol with Fertility and Sterility
FIGURE 1 Ultrasound image of monochorionic, monoamniotic intrauterine twin pregnancy with two fetal heartbeats (case 1).
Esfandiari. Late maternal age and monozygotic twinning. Fertil Steril 2009.
buserelin acetate (Suprefact, Aventis) twice daily starting on cycle day 3, and recombinant FSH (300 IU Puregon; Organon) starting on day 3 until day 11 of her menstrual cycle. Human chorionic gonadotrophin (10,000 IU; Organon) was administered to trigger the release of the oocytes. Thirty-six hours later 20 oocytes were retrieved by ultrasound-guided needle aspiration. Five oocytes were inseminated 3 hours after the retrieval, and the remaining 15 oocytes underwent ICSI. After removal of cumulus cells with hyaluronidase (Sigma Chemical), 12 of the oocytes were mature at metaphase II, and 3 were immature at metaphase I. All 12 mature oocytes were injected. Semen analysis showed a volume of 1.5 mL, sperm concentration of 110 106/mL, and 55% forward progressive motility. The morphology according to World Health Organization (1999) criteria was 20%, with moderate sperm agglutination. The sperm parameters after processing with density gradient were volume of 0.5 mL with a concentration of 60 106/ mL, 92% motility, and 30% normal morphology. 1168.e10
Eighteen hours after insemination, four of the IVF oocytes were normally fertilized (2PN) zygotes, and one zygote was 1PN. Out of 12 ICSI oocytes injected, 10 were normally fertilized (2PN) zygotes, and 2 were 1PN. The fertilized oocytes were placed in microdrops of P1 (Irvine Scientific) with 10% synthetic serum substitute under mineral oil (Sigma Chemical) and left in culture for an additional 2 days. All normally fertilized oocytes cleaved and resulted in nine eight-cell grade 1 embryos, three eight-cell grade 2, and one six-cell grade 1. One eight-cell grade 2, one six-cell grade 2, and two four-cell grade 2 embryos resulted from 1PN fertilization. After undergoing laser-assisted hatching, seven high-grade embryos (three from IVF and four from ICSI) were transferred to the patient. The embryos were transferred using a Cook catheter (Cook Australia, Queensland, Australia). Two weeks after ET the patient had a serum b-hCG level of 990 IU/L. At 7 weeks’ gestation, ultrasound examination revealed an appropriately sized monochorionic, diamniotic twin intrauterine pregnancy with two fetal heartbeats. The patient delivered twin boys at 26 weeks. They weighed 800 g each; both were in the hospital for 3 months. DISCUSSION It is known that the risk of monozygotic twins in women undergoing assisted reproduction is more than twofold that of the normal population. The exact mechanism(s) leading to MZT is still controversial, and the literature provides multiple theories (9). Considering the elevated morbidity and mortality involved with monozygotic twin pregnancies, the ability to identify predisposing factors would be highly beneficial. Monozygotic twin pregnancies have always been thought to be independent of genetics. However, case studies have reported a high frequency of monozygosity in certain families. Shapiro et al. (5) reported on 10 different families with multiple pairs of MZT, a frequency far exceeding the incidence of the general population. Parisi et al. (6) described a case of monozygotic twin parents. Pedigree analysis of both families suggested a monogenic mode of inheritance transmitted by both parents, but with low penetrance. Conversely, Litchenstein et al. (10) found no evidence of a paternal effect on monozygotic twinning. The influence of ART on monozygotic twinning is uncertain, mainly owing to the overall low incidence of monozygotic twins. Ovarian stimulation may play a role by delaying implantation and hardening of the zona. It is also possible that some oocytes have an inherent potential to undergo splitting upon fertilization. Ovarian stimulation may simply boost this potential. In older patients the chance of transferring a ‘‘splitting-prone’’ oocyte is more likely because of the higher number of transferred embryos. Suboptimal in vitro culture conditions have been considered another possible etiology behind monozygotic twin1168.e11
Esfandiari et al.
ning. The artificial culture conditions are thought to cause an insult to the early embryo by disrupting communication between blastomeres, resulting in an independent formation of two separate blastocysts (7). Another theory on in vitro culture conditions is that prolonged exposure to the artificial medium leads to a possible change of the ZP, which in turn could lead to increased fragility and predisposition to fractures (3). Micromanipulation of the ZP has been speculated to cause MZT. With ICSI, a spermatozoon is directly injected into the oocyte cytoplasm. The technique greatly reduces the number of sperm needed for IVF and has resulted in successful pregnancy in many couples, including those with severe male factor infertility. Injection of a sperm damages the zona by making a 6–8-mm diameter opening in the zona. Assisted hatching (AH) is another technique that involves micromanipulation of the ZP. Assisted hatching involves chemical, mechanical, or laser thinning of the ZP of a fertilized embryo. This is done to facilitate embryo hatching and enhance the probability of implantation (7). Perhaps during the process of AH the artificial breech in the ZP promotes the chance of subsequent herniation of blastomeres through the hole in the zona when the blastocyst is expanding. The event is thought to result in a mechanical splitting of the blastocyst and MZT (11). Although many studies support the association between micromanipulation of the ZP and MZT, there are studies that conclude there is no association. A retrospective study by Schachter et al. (4) found that the rate of MZT was consistently increased, irrespective of treatment modality or micromanipulation. Recently Sills et al. (12), in a review of twin pregnancies including 23 MZTs, reported that among IVF study patients the frequency of MZT was not statistically different between zona-manipulated and zona-intact subgroups. Zona opening in AH forms a puncture with a diameter between 20 and 30 mm, which is several times bigger than the defect caused by the needle during ICSI. Surprisingly, in a recent meta-analysis, ICSI was more highly correlated with incidence of MZT than was AH (MZT rate of 1.0% vs. 0.7%). A major limitation of all the studies reported in the literature is the low incidence of MZT, even in the ART setting. Therefore, a large study with adequate power would be beneficial. Transferring embryos at the blastocyst stage is believed to result in an increased MZT rate (13, 14). This is in contrast to the general consensus that blastocyst culture facilitates selection of the one or two best-quality embryos for transfer, thereby practically eliminating the possibility of high-order multiple pregnancies. Several mechanisms have been suggested to explain the observed increased MZT rate after blastocyst transfer, including extended culture time and culture media composition (14). Maternal age is an independent factor determining the success of ART treatment, and advanced female age is associated with a decline in success rate. Oocyte number, quality,
Late maternal age and monozygotic twinning
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and resulting embryo quality after fertilization are involved in poor success rates in older patients. Because of increased aneuploidy rate and lower quality, the number of transferred embryos in older aged patients is suggested to be four in 38- and 39-year-olds, and up to five embryos in R40-yearold patients (15). Advanced maternal age (>35 years) is associated with an increased risk of dizygotic twins. The prevalence of naturally occurring twin births has increased recently because of the trend for delaying childbearing. There is a gradual decrease in the average thickness of the ZP with increasing age. This naturally irregularly thin ZP may affect the hatching process in the same manner as AH if, for instance, the blastocyst protrudes at more than one place due to multiple sites of zona lysis (2). Monozygotic twin pregnancies have a known higher risk of complications compared with singleton and dizygotic twin pregnancies. Complications include a higher percentage of premature delivery, growth discordance, and developmental anomalies (16, 17). In addition, twin-to-twin transfusion syndrome is a serious disorder unique to multiples that share a placenta. Twin-to-twin transfusion syndrome occurs in 5%– 38% of monochorionic twins and in severe form has a 60%– 100% fetal or neonatal mortality rate. Mild-to-moderate twin-to-twin transfusion syndrome is frequently associated with premature delivery (18). This complication occurs when there is an arteriovenous connection between the blood vessels in the shared placenta of the two fetuses. The recipient twin is the one receiving more blood flow, and the donor twin is one who receives too little (19). The several possible complications associated with monozygosity lead to an elevated perinatal mortality. In conclusion, although the exact mechanism(s) by which the two reported patients in the present case series developed MZT is not known, they shared multiple possible risk factors. Although neither of the patients had a history of monozygotic twinning, both patients were of advanced maternal age (44 and 43 years old), with history of secondary infertility. Both patients underwent assisted reproduction procedures, including high-dose gonadotropin stimulation, embryo culture, and, in case 2, ICSI plus AH. Because of the low incidence of MZT in both the natural and assisted reproductive settings, an adequate study investigating the exact etiology of this condition is difficult. It is possible that the incidence of MZT may actually be underestimated. Many dichorionic, diamniotic twins may be monozygotic but reported as dizygotic. In addition, with spontaneous conception in the absence of early ultrasound, many cases of the vanishing twin may either be missed or not reported. At this time we can only speculate about potential risk factors, which may be important to
Fertility and Sterility
keep in mind when counseling older patients undergoing IVF. Acknowledgment: The authors thank Roger Stronell, M.D., for providing the ultrasound images.
REFERENCES 1. Kerin JF, Warnes GM, Quinn PJ, Jeffrey R, Kirby C, Matthews CD, et al. Incidence of multiple pregnancy after in-vitro fertilization and embryo transfer. Lancet 1983;2:537–40. 2. Abusheika N, Salha O, Sharma V, Brinsden P. Monozygotic twinning and IVF/ICSI treatment: a report of 11 cases and review of literature. Hum Reprod Update 2000;6:396–403. 3. Edwards RG, Mettler L, Walters DE. Identical twins and in vitro fertilization. J In Vitro Fertil Embryo Trans 1986;3:114–7. 4. Schachter M, Raziel A, Friedler S, Strassburger D, Bern O, Ron-El R. Monozygotic twinning after assisted reproductive techniques: a phenomenon independent of micromanipulation. Hum Reprod 2001;16:1264–9. 5. Shapiro LR, Zemek L, Shulman MJ. Genetic etiology for monozygotic twinning. Birth Defects Orig Artic Ser 1978;14:219–22. 6. Parisi P, Gatti M, Prinzi G, Caperna G. Familial incidence of twinning. Nature 1983;304:626–8. 7. Aston KI, Peterson CM, Carrell DT. Monozygotic twinning associated with assisted reproductive technologies: a review. Reproduction 2008;136:377–86. 8. Yovich JL, Stanger JD, Grauaug A, Barter RA, Lunay G, Dawkins RL, et al. Monozygotic twins from in vitro fertilization. Fertil Steril 1984;41:833–7. 9. 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;15:45–55. 10. Lichtenstein P, Kall€en B, K€oster M. No paternal effect on monozygotic twinning in the Swedish Twin Registry. Twin Res 1998;1:212–5. 11. Talansky BE, Gordon JW. Cleavage characteristics of mouse embryos inseminated and cultured after zona pellucida drilling. Gamete Res 1988;31:277–87. 12. Sills ES, Moomjy M, Zaninovic N, Veeck LL, McGee M, Palermo GD, et al. Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF. Hum Reprod 2000;15:890–5. 13. Chang HJ, Lee JR, Jee BC, Suh CS, Kim SH. Impact of blastocyst transfer on offspring sex ratio and the monozygotic twinning rate: a systematic review and meta-analysis. Fertil Steril 2009;91:2381–90. 14. 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;79:503–6. 15. Stern JE, Goldman MB, Hatasaka H, Mackenzie TA, Surrey ES, Racowsky C. Society for Assisted Reproductive Technology (SART) Writing Group. Optimizing the number of cleavage stage embryos to transfer on day 3 in women 38 years of age and older: a Society for Assisted Reproductive Technology database study. Fertil Steril 2009;91:767–76. 16. Sebire NJ, Snijders RJ, Hughes K, Sepulveda W, Nicolaides KH. The hidden mortality of monochorionic twin pregnancies. Br J Obstet Gynaecol 1997;104:1203–7. 17. Maier RF, Bialobrzeski B, Gross A, Vogel M, Dudenhausen JW, Obladen M. Acute and chronic fetal hypoxia in monochorionic and dichorionic twins. Obstet Gynecol 1995;86:973–7. 18. Zach, T, Barsoom MJ. Twin-to-twin transfusion syndrome. http://emedi cine.medscape.com/article/271752 2007. Last accessed May 1, 2009. 19. Robertson EG, Neer KJ. Placental injection studies in twin gestation. Am J Obstet Gynecol 1983;147:170–4.
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Toronto Centre for Advanced Reproductive Technology, and b Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada
Objective: To report two cases of successful monozygotic twin pregnancies in women undergoing infertility treatment and to review possible etiologic factors. Design: Case report and review of the literature. Setting: University of Toronto–affiliated infertility clinic. Patient(s): A 43-year-old woman and a 44-year-old woman with history of secondary infertility. Intervention(s): In vitro fertilization and ET. Main Outcome Measure(s): Monozygotic twin pregnancy. Result(s): Delivery of two sets of monozygotic twins. Conclusion(s): The only identified potential risk factors for monozygotic twins are maternal age and assisted reproductive technologies. Both patients reported here were aged >40 years and underwent assisted reproduction procedures, including high-dose gonadotropin stimulation, embryo culture, and, in case 2, intracytoplasmic sperm injection plus assisted hatching. (Fertil Steril 2009;92:1168.e9–e12. 2009 by American Society for Reproductive Medicine.) Key Words: Monozygotic twinning, maternal age, IVF
Today it is common for women in developed nations to delay childbearing until the fourth decade of life. As fertility declines with age, more couples turn to assisted reproductive technologies (ART) to conceive. Since the first IVF baby born in 1978, this procedure has granted many infertile couples an opportunity for pregnancy, often resulting in an increased incidence of multiple pregnancies (1). At present approximately one third of IVF babies are twins, mostly because more than one embryo is transferred to the uterus to maximize the chance of a successful pregnancy. Dizygotic twins are a known risk of multiple ovulations and multiple embryo transfer. However, data on the etiology of monozygotic twinning (MZT) is limited. Monozygotic twinning is a relatively rare event and is estimated to occur in 0.4% of in-vivo conception (2), compared with approximately 1.5% of all clinical pregnancies after ovulation induction with gonadotropins (3). Monozygotic twinning is Received March 10, 2009; revised May 13, 2009; accepted May 27, 2009; published online July 15, 2009. N.E. has nothing to disclose. M.K. has nothing to disclose. H.B. has nothing to disclose. P.C. has nothing to disclose. L.G. has nothing to disclose. R.F.C. has nothing to disclose. Reprint requests: Navid Esfandiari, D.V.M., Ph.D., H.C.L.D., Toronto Centre for advanced Reproductive Technology, 210-150 Bloor Street West, Toronto, ON M5S 2X9, Canada (FAX: 416-972 0036; E-mail: navid. [email protected]).
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defined according to the stage of development when division of the fertilized ovum occurs. Biamniotic, dichorionic twins occur when division of the embryonic cell mass occurs earlier than 72 hours after fertilization. If division occurs between days 4 to 8 after the inner mass has formed, biamniotic, monochorionic twins will result. Division after day 8 will lead to a monoamniotic, monochorionic twin pregnancy (4). The process of monozygotic twinning is speculated to begin with the protrusion of some trophectoderm cells through a small opening in the zona pellucida (ZP). Some of the cell mass may then break off in the uterus to form a twin pregnancy. Several factors associated with MZT include family history (5, 6), ovarian stimulation (4), the use of intracytoplasmic sperm injection (ICSI), in vitro embryo culture conditions (3, 7), ZP manipulation (7), stage of embryo development at the time of transfer, and maternal age. The first monozygotic pregnancy resulting from IVF was reported by Yovich et al. in 1984 (8). This case study described the outcome of monochorionic, diamniotic twin males. Since the report of this case, there have been many additions to the literature describing similar cases. However, factors that affect the frequency of MZT have been poorly characterized. Monozygotic twinning is considered to be a higher-risk pregnancy that requires additional surveillance compared with dizygotic twinning. The ability to identify risk factors is
Fertility and Sterility Vol. 92, No. 3, September 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2009.05.085
very important to provide adequate counseling and care to the patient. The present case reports describe MZT in two infertility patients with advanced maternal age. CASE REPORTS Case 1 A 43-year-old gravida 1, para 0 patient was referred to our center after 1 year of secondary infertility. She had no prior tubal risk factor and normal results on hysterosalpingogram before her ectopic pregnancy. For her fourth IVF cycle in October 2005 she received a modified microdose flare protocol using letrozole (2.5 mg; Novartis, Dorval, QC, Canada) from day 3 to day 7 of her menstrual cycle, buserelin acetate (Suprefact 0.05 mL SC twice daily; Aventis Pharma, Laval, QC, Canada) starting on day 3, and recombinant FSH (Puregon 300 IU daily; Organon, Scarborough, ON, Canada) starting on day 3. On day 15 of her cycle, 10,000 IU of hCG (Organon) was given. Thirty-six hours later, nine oocytes were retrieved by ultrasound-guided needle aspiration. All oocytes were inseminated 3 hours after the retrieval. Semen analyses revealed a volume of 0.6 mL with a concentration of 34 106/mL and 60% forward progressive motility. The morphology according to World Health Organization (1999) criteria was 18%, with moderate sperm agglutination. The sperm parameters after processing with density gradient was a volume of 0.4 mL with a concentration of 16 106/mL, 98% motility, and 28% normal morphology. Eighteen hours after insemination, only four oocytes were normally fertilized and one oocyte fertilized with 1 pronucleus (PN). The fertilized oocytes were placed in microdrops of P1 (Irvine Scientific, Santa Ana, CA) with 10% synthetic serum substitute under mineral oil (Sigma Chemical, St. Louis, MO) and left in culture for an additional 2 days. All fertilized oocytes cleaved and resulted in one six-cell grade 1 embryo, one four-cell grade 1, one three-cell grade 2, one two-cell grade 2, and one five-cell grade 2 from 1PN fertilization. The grading system used was based on a scale of 1–4, with grade 1 representing highest quality. These five IVF embryos were transferred using a Tomcat catheter (Meditech, Montreal, QC, Canada). Two weeks after ET the patient had a serum b-hCG level of 391 IU/L and 933 IU/L 2 days later. At 8 weeks’ gestation, an ultrasound examination revealed an appropriately sized monochorionic, monoamniotic intrauterine twin pregnancy (Fig. 1) with two fetal heartbeats. The patient delivered twin girls by cesarian section at 34 weeks’ gestation, weighing 2,100 g and 2,300 g, respectively. Hemangioma was observed on the face of one newborn. Case 2 A 44-year-old gravida 1, para 1 patient was referred to our center with 3 years of unexplained secondary infertility. She had mild endometriosis confirmed by laparoscopy. After three failed IVF/ICSI cycles, she began her fourth attempt in April 2006. She received a microdose flare protocol with Fertility and Sterility
FIGURE 1 Ultrasound image of monochorionic, monoamniotic intrauterine twin pregnancy with two fetal heartbeats (case 1).
Esfandiari. Late maternal age and monozygotic twinning. Fertil Steril 2009.
buserelin acetate (Suprefact, Aventis) twice daily starting on cycle day 3, and recombinant FSH (300 IU Puregon; Organon) starting on day 3 until day 11 of her menstrual cycle. Human chorionic gonadotrophin (10,000 IU; Organon) was administered to trigger the release of the oocytes. Thirty-six hours later 20 oocytes were retrieved by ultrasound-guided needle aspiration. Five oocytes were inseminated 3 hours after the retrieval, and the remaining 15 oocytes underwent ICSI. After removal of cumulus cells with hyaluronidase (Sigma Chemical), 12 of the oocytes were mature at metaphase II, and 3 were immature at metaphase I. All 12 mature oocytes were injected. Semen analysis showed a volume of 1.5 mL, sperm concentration of 110 106/mL, and 55% forward progressive motility. The morphology according to World Health Organization (1999) criteria was 20%, with moderate sperm agglutination. The sperm parameters after processing with density gradient were volume of 0.5 mL with a concentration of 60 106/ mL, 92% motility, and 30% normal morphology. 1168.e10
Eighteen hours after insemination, four of the IVF oocytes were normally fertilized (2PN) zygotes, and one zygote was 1PN. Out of 12 ICSI oocytes injected, 10 were normally fertilized (2PN) zygotes, and 2 were 1PN. The fertilized oocytes were placed in microdrops of P1 (Irvine Scientific) with 10% synthetic serum substitute under mineral oil (Sigma Chemical) and left in culture for an additional 2 days. All normally fertilized oocytes cleaved and resulted in nine eight-cell grade 1 embryos, three eight-cell grade 2, and one six-cell grade 1. One eight-cell grade 2, one six-cell grade 2, and two four-cell grade 2 embryos resulted from 1PN fertilization. After undergoing laser-assisted hatching, seven high-grade embryos (three from IVF and four from ICSI) were transferred to the patient. The embryos were transferred using a Cook catheter (Cook Australia, Queensland, Australia). Two weeks after ET the patient had a serum b-hCG level of 990 IU/L. At 7 weeks’ gestation, ultrasound examination revealed an appropriately sized monochorionic, diamniotic twin intrauterine pregnancy with two fetal heartbeats. The patient delivered twin boys at 26 weeks. They weighed 800 g each; both were in the hospital for 3 months. DISCUSSION It is known that the risk of monozygotic twins in women undergoing assisted reproduction is more than twofold that of the normal population. The exact mechanism(s) leading to MZT is still controversial, and the literature provides multiple theories (9). Considering the elevated morbidity and mortality involved with monozygotic twin pregnancies, the ability to identify predisposing factors would be highly beneficial. Monozygotic twin pregnancies have always been thought to be independent of genetics. However, case studies have reported a high frequency of monozygosity in certain families. Shapiro et al. (5) reported on 10 different families with multiple pairs of MZT, a frequency far exceeding the incidence of the general population. Parisi et al. (6) described a case of monozygotic twin parents. Pedigree analysis of both families suggested a monogenic mode of inheritance transmitted by both parents, but with low penetrance. Conversely, Litchenstein et al. (10) found no evidence of a paternal effect on monozygotic twinning. The influence of ART on monozygotic twinning is uncertain, mainly owing to the overall low incidence of monozygotic twins. Ovarian stimulation may play a role by delaying implantation and hardening of the zona. It is also possible that some oocytes have an inherent potential to undergo splitting upon fertilization. Ovarian stimulation may simply boost this potential. In older patients the chance of transferring a ‘‘splitting-prone’’ oocyte is more likely because of the higher number of transferred embryos. Suboptimal in vitro culture conditions have been considered another possible etiology behind monozygotic twin1168.e11
Esfandiari et al.
ning. The artificial culture conditions are thought to cause an insult to the early embryo by disrupting communication between blastomeres, resulting in an independent formation of two separate blastocysts (7). Another theory on in vitro culture conditions is that prolonged exposure to the artificial medium leads to a possible change of the ZP, which in turn could lead to increased fragility and predisposition to fractures (3). Micromanipulation of the ZP has been speculated to cause MZT. With ICSI, a spermatozoon is directly injected into the oocyte cytoplasm. The technique greatly reduces the number of sperm needed for IVF and has resulted in successful pregnancy in many couples, including those with severe male factor infertility. Injection of a sperm damages the zona by making a 6–8-mm diameter opening in the zona. Assisted hatching (AH) is another technique that involves micromanipulation of the ZP. Assisted hatching involves chemical, mechanical, or laser thinning of the ZP of a fertilized embryo. This is done to facilitate embryo hatching and enhance the probability of implantation (7). Perhaps during the process of AH the artificial breech in the ZP promotes the chance of subsequent herniation of blastomeres through the hole in the zona when the blastocyst is expanding. The event is thought to result in a mechanical splitting of the blastocyst and MZT (11). Although many studies support the association between micromanipulation of the ZP and MZT, there are studies that conclude there is no association. A retrospective study by Schachter et al. (4) found that the rate of MZT was consistently increased, irrespective of treatment modality or micromanipulation. Recently Sills et al. (12), in a review of twin pregnancies including 23 MZTs, reported that among IVF study patients the frequency of MZT was not statistically different between zona-manipulated and zona-intact subgroups. Zona opening in AH forms a puncture with a diameter between 20 and 30 mm, which is several times bigger than the defect caused by the needle during ICSI. Surprisingly, in a recent meta-analysis, ICSI was more highly correlated with incidence of MZT than was AH (MZT rate of 1.0% vs. 0.7%). A major limitation of all the studies reported in the literature is the low incidence of MZT, even in the ART setting. Therefore, a large study with adequate power would be beneficial. Transferring embryos at the blastocyst stage is believed to result in an increased MZT rate (13, 14). This is in contrast to the general consensus that blastocyst culture facilitates selection of the one or two best-quality embryos for transfer, thereby practically eliminating the possibility of high-order multiple pregnancies. Several mechanisms have been suggested to explain the observed increased MZT rate after blastocyst transfer, including extended culture time and culture media composition (14). Maternal age is an independent factor determining the success of ART treatment, and advanced female age is associated with a decline in success rate. Oocyte number, quality,
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and resulting embryo quality after fertilization are involved in poor success rates in older patients. Because of increased aneuploidy rate and lower quality, the number of transferred embryos in older aged patients is suggested to be four in 38- and 39-year-olds, and up to five embryos in R40-yearold patients (15). Advanced maternal age (>35 years) is associated with an increased risk of dizygotic twins. The prevalence of naturally occurring twin births has increased recently because of the trend for delaying childbearing. There is a gradual decrease in the average thickness of the ZP with increasing age. This naturally irregularly thin ZP may affect the hatching process in the same manner as AH if, for instance, the blastocyst protrudes at more than one place due to multiple sites of zona lysis (2). Monozygotic twin pregnancies have a known higher risk of complications compared with singleton and dizygotic twin pregnancies. Complications include a higher percentage of premature delivery, growth discordance, and developmental anomalies (16, 17). In addition, twin-to-twin transfusion syndrome is a serious disorder unique to multiples that share a placenta. Twin-to-twin transfusion syndrome occurs in 5%– 38% of monochorionic twins and in severe form has a 60%– 100% fetal or neonatal mortality rate. Mild-to-moderate twin-to-twin transfusion syndrome is frequently associated with premature delivery (18). This complication occurs when there is an arteriovenous connection between the blood vessels in the shared placenta of the two fetuses. The recipient twin is the one receiving more blood flow, and the donor twin is one who receives too little (19). The several possible complications associated with monozygosity lead to an elevated perinatal mortality. In conclusion, although the exact mechanism(s) by which the two reported patients in the present case series developed MZT is not known, they shared multiple possible risk factors. Although neither of the patients had a history of monozygotic twinning, both patients were of advanced maternal age (44 and 43 years old), with history of secondary infertility. Both patients underwent assisted reproduction procedures, including high-dose gonadotropin stimulation, embryo culture, and, in case 2, ICSI plus AH. Because of the low incidence of MZT in both the natural and assisted reproductive settings, an adequate study investigating the exact etiology of this condition is difficult. It is possible that the incidence of MZT may actually be underestimated. Many dichorionic, diamniotic twins may be monozygotic but reported as dizygotic. In addition, with spontaneous conception in the absence of early ultrasound, many cases of the vanishing twin may either be missed or not reported. At this time we can only speculate about potential risk factors, which may be important to
Fertility and Sterility
keep in mind when counseling older patients undergoing IVF. Acknowledgment: The authors thank Roger Stronell, M.D., for providing the ultrasound images.
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