twinning Braulio Peramo, M.D., Elisabetta Ricciarelli, M.D., Jorge M. Cuadros-Fernández, Ph.D., Eva Huguet, Ph.D., and Eleuterio Rodrigo Hernández, M.D., Ph.D. Clı́nica de Reproducción Asistida FIV-Madrid and Instituto de Bioquı́mica, CSIC, Madrid, Spain
Objective: To report two cases of monozygotic twinning after IVF-blastocyst transfer.
The reported prevalence of multiple gesta- CASE REPORTS
tions in IVF-ET is approximately 30% (1), and it is well known that multiple births are asso- A 35-year-old woman (gravida 1, para 0) ciated with a high risk of maternal and fetal with a 3-year history of unexplained infertility morbidity and mortality. In general, multiple and a 34-year-old woman (gravida 0, para 0) pregnancies in IVF result from the transfer of with a 4-year history of tubal infertility under- ⬎3 embryos (2). Several strategies have been went IVF-ET. Neither patient had a history of familial twinning. Both patients received the proposed to decrease the number of multiple so-called “long protocol” for ovulation induc- pregnancies. One is to limit the number of tion. Briefly, pituitary suppression was embryos transferred to two, and the other is to achieved using GnRH agonist (Procrin; Abbott increase pregnancy rates by transferring em- Laboratories, Madrid, Spain). Once down-reg- bryos at the blastocyst stage (3). Although ulation was confirmed, follicular development Received February 23, these strategies may reduce the rate of multiple was stimulated with gonadotropins (Neoferti- 1999; revised and pregnancies in IVF, embryos derived after ma- norm; Serono Laboratories, Madrid, Spain). accepted June 30, 1999. nipulation of the zona pellucida (ZP) are more Reprint requests: E. R. When at least two to three follicles attained a Hernández, M.D., Ph.D., likely to cause multiple pregnancies. For in- size of ⬎18 mm in diameter, 10,000 IU of hCG Clı́nica de Reproducción stance, monozygotic twinning has been related (Profasi; Serono Laboratories) was adminis- Asistida FIV-Madrid, C/ to subzonal insemination (SUZI), intracyto- Álvarez de Baena 4, tered. Thirty-four hours later, oocytes were re- 28006 Madrid, Spain plasmic sperm injection (ICSI), and assisted trieved by ultrasound (US) guided puncture. (FAX: 34-915-610-700; hatching (4). E-mail: ehernandezm Oocytes were washed in Ham’s F-10 me- @meditex.es). To our knowledge, we report the first cases dium (Life Technologies, Paisley, Scotland), of monozygotic twinning related to blastocyst supplemented with penicillin (10,000 IU/mL) 0015-0282/99/$20.00 PII S0015-0282(99)00412-4 transfer without ZP manipulation. and streptomycin (10,000 g/mL), and then
1116 placed in a culture dish containing 3 mL of chemically Patient number 2 received two blastocysts. A positive defined commercial medium: IVF Medium (Medi-Cult, level of -hCG (66 IU/L) was observed 12 days after transfer Copenhagen, Denmark) or IVF-50 (Scandinavian IVF Sci- and increased 2 and 4 days later (126 and 218 IU/L). At 28 ence, Gothenborg, Sweden). Retrieved oocytes were fertil- days after retrieval, transvaginal US showed a single gesta- ized by conventional IVF with 50,000 spermatozoa per mil- tional sac containing two embryos (with positive heartbeats), liliter added to the culture media 3–5 hours after retrieval. both with a crown-rump length of 4 mm. At 7 weeks’ Fertilization occurred 18 –20 hours after insemination gestation, the lengths were 13 mm and 12 mm. Each embryo (day 1). Those oocytes with two pronuclei and two polar had its own amniotic cavity. Amniocentesis at 14 weeks’ bodies were maintained for another 24 hours in the same gestation (by patient request) revealed a karyotype of 46,XX culture conditions. Triploid embryos were discarded. for both embryos. During the pregnancy, twin-twin transfu- sion syndrome was observed. The patient was hospitalized On day 2, the embryos were classified based on the for meticulous monitoring of the fetuses. Because the ho- number of blastomeres and percentage of cytoplasmic frag- meostatic balance between the fetuses deteriorated over the ments. After grading, the zygotes were transferred to a new gestational course, two girls (1,250 and 1,150 g) were de- culture dish containing 3 mL of S2-20 medium (Scandina- livered by cesarean section at 28 weeks’ gestation. vian IVF Science) and were cultured in a group until the embryo reached the blastocyst stage. Progression and quality were observed daily. The blastocysts characterized by an DISCUSSION expanded cavity and an eccentric inner cell mass (ICM) were considered good blastocysts and were scored as grade BG1 We are currently using a defined, commercially available or BG2. The BG1/BG2 blastocysts were transferred mostly culture medium (S2-20) to maintain the growth of human at days 5– 6 after retrieval. zygotes to the blastocyst stage in a sequential culture. Be- P (Progeffik; Effik Laboratories, Madrid, Spain) at 600 tween January and October 1998, 94 blastocysts were trans- mg/d was given throughout the luteal phase. If a gestational ferred to 41 patients (2.3 embryos per transfer), and only 2 sac with a positive heartbeat was detected by US, P was patients (4.8%) developed monozygotic twinning. slowly decreased and discontinued by week 12. A search of the existing body of literature on monozy- Institutional review board approval was not required be- gotic twinning after blastocyst transfer using the MEDLINE cause the interventions performed in this study were not computer database failed to identify any other case. To the considered human experimentation, and all them are com- best of our knowledge, this is the first report of monozygotic monly used in diagnostic and therapeutic procedures. twinning after blastocyst transfer without ZP manipulation. In patient number 1, two blastocysts were transferred at Although monozygotic twinning in IVF has been related day 5 after retrieval. A positive level of -hCG (658 IU/L) to SUZI, ICSI, and assisted hatching, we do not know at was observed 12 days after transfer and increased 2 days which moment, or by which mechanism, the ICM segre- later (1,248 IU/L). Transvaginal US (28 days after retrieval) gates. However, we think that (apart from genetic origin) showed two gestational sacs, one with an embryo with a some trauma may occur during transfer of the embryos to the crown-rump length of 6 mm and the other containing two uterus. Other possible mechanisms may include changes in embryos with crown-rump lengths of 5 mm and 3 mm. All of the ZP or in the intercellular unions by the culture media. the embryos had a heartbeat. The last two embryos shared References the same amniotic cavity. At 8 gestational weeks, transvag- 1. World Collaborative Report on In Vitro Fertilization. Preliminary data inal US examination showed two gestational sacs only, one for 1995. J Assist Reprod Genet 1997;14:251S– 65S. 2. Geva E, Amit A, Lerner-Geva L, Lessing JB. Embryo transfer and with a single embryo with a crown-rump length of 23 mm, multiple gestation. How many transfers are too many? Hum Reprod and the other with an embryo of 23 mm, plus a vestige of an 1998;13:2988 –9. 3. Coetsier T, Dhont M. Avoiding multiple pregnancies in in-vitro fertili- embryo (seen as a hyperechogenic image of 5 mm). The zation: who’s afraid of single embryo transfer? Hum Reprod 1998;13: course of this pregnancy was normal, and two children were 2663– 4. 4. Alikani M, Noyes N, Cohen J, Rosenwaks Z. Monozygotic twinning in born at 37 weeks’ gestation by cesarean section (weights of the human is associated with the zona pellucida architecture. Hum 2,850 g and 2,760 g, respectively). Reprod 1994;9:1318 –21.
Live Birth After Transfer of A Single Euploid Vitrified-Warmed Blastocyst According To Standard Timing vs. Timing As Recommended by Endometrial Receptivity Analysis
IN VITRO MATURATION OF OOCYTES: A BREAKTHROUGH FOR TREATING INFERTILITY IN INACTIVATING MUTATION OF THE LUTEINIZING HORMONE/CHORIOGONADOTROPIN RECEPTOR
