Two cases of dizygotic triplet pregnancy following conventional in vitro fertilization

Abstract

The mechanism of monozygotic multifetal pregnancy and its association with assisted reproductive technology are uncertain. This report presents two cases of dizygotic triplet pregnancy after the transfer of three embryos in utero. The incidence of monozygotic twinning following assisted conception procedures is higher than in the general population. Zonal manipulation may be a major factor in the increased incidence of monozygotic twinning. As both cases followed the conventional in vitro fertilization (IVF), the reason for these monozygotic twinnings might be because of the changes in the zona pellucida in in vitro conditions. In one case, fetuses developed into severe twin‐to‐twin transfusion syndrome (TTTS), resulting in one intrauterine fetal death at 26 weeks gestation. (Reprod Med Biol 2003; 2: 41–44)

INTRODUCTION

IT IS WELL known that assisted reproductive technique is complicated by the increase in the rate of multifetal pregnancies. The incidence of multiple gestation in in vitro fertilization (IVF) is approximately 30% according to a previous report with higher morbidity and mortality. ¹ As most multifetal pregnancies in IVF have been shown to be caused by the fertilization of multiple ova the number of embryos transferred has been limited. ² , ³ Although the limitation of the embryos transferred has undoubtedly decreased the incidence of multifetal pregnancy, the risk to the fetus because of monozygotic multifetal pregnancy in IVF has been brought to light. We report here two cases of dizygotic triplet pregnancy; one case of monozygotic twins and another fetus after IVF.

CASE REPORT

Case 1

A 34‐YEAR‐OLD WOMAN (gravida 0, para 0) without a history of familiar disposition to twinning and having polycystic ovaries, was infertile for 6 years. The stimulation protocol was as described elsewhere. ⁴ In brief, long protocol pituitary suppression was performed by using the gonadotropin‐releasing hormone (GnRH) agonist, nafarelin acetate (Nasanyl; Yamanouchi Pharmaceutical, Tokyo, Japan). Once the downregulation of the pituitary was confirmed, the follicular development was stimulated with human menopausal gonadotropin (hMG ‘Nikken’, Nikken Chemicals, Tokyo, Japan), which was injected daily for 7 days, from the fourth cycle day (1350 IU totally). The leading follicle reached a diameter of 18 mm and the plasma estradiol concentration increased to 2158 pmol/L on the twelfth cycle day. Thirty‐five hours later, 10 000 IU of human chorionic gonadotropin (hCG, Gonatrophin; Teikoku Hormone Mfg, Tokyo, Japan) was administered intramuscularly, then the transvaginal collection succeeded in collecting 17 oocytes. The retrieved oocytes were fertilized by using conventional IVF techniques with 150 000 motile spermatozoa per milliliter added to the culture media 3 h after retrieval. Fertilization of nine oocytes occurred 18 h after insemination (day 1). These oocytes with two pronuclei and two polar bodies were maintained for 24 h. Oocytes and embryos were cultured in Human Tubal Fluid Media (HTF Medium; Irvin Scientific, Santa Ana, CA, USA) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific, Santa Ana, CA, USA) in an atmosphere of 5% O₂. On day 2, the embryos were classified based on the number of blastomeres and percentage of cytoplasmic fragments. After grading, three embryos (grade 1 at the 4‐cell stage, grade 2 at the 4‐cell stage, and grade 4 at the 5‐cell stage) were transferred to the uterus by using a Phycon IVF Catheter (Fuji System Ltd, Tokyo, Japan). ⁵ The luteal phase was supported with 50 mg progesterone (Ruteum Injection; Teikoku Hormone Mfg, Tokyo, Japan) i.m. per day for 14 days.

Two weeks after the embryo transfer, the patient's semiquantitative β‐HCG test was positive. The transvaginal ultrasound scan (21 days after retrieval) showed the two gestational sacs in her uterus. At 9 weeks gestation, the ultrasound scan showed three viable fetuses in three amniotic cavies, two of which were contained in the same gestational sac. Therefore, dizygotic triplet pregnancy was diagnosed.

At 26 weeks of gestation, the patient was admitted to our hospital for management of her pregnancy. An emergency Cesarean section was performed because of an abruptio placentae at 34 gestational weeks. The birth weights were 2060 g (girl), 1648 g and 1384 g (monozygotic girls). Examination of the placentae showed a dichorionic triplet pregnancy.

Case 2

A 36‐year‐old woman (gravida 0, para 0) without familial tendency to twinning, having a past history of hyperprolactinemia, was infertile for 4 years. She was treated for IVF. The stimulation protocol was the same as in Case 1. The pituitary suppression was undertaken by nafarelin acetate, and once the downregulation of the pituitary was confirmed, the follicular development was stimulated with hMG, which was injected daily for 7 days, starting on the eighth cycle day (1200 IU totally). The leading follicle reached a diameter of 18 mm and plasma estradiol concentration of 4487 pmol/L on the 14th cycle day. Thirty‐five hours later, 10 000 IU of hCG i.m. was administered before a transvaginal collection succeeded in collecting 18 oocytes. The retrieved oocytes were fertilized with 150 000 motile spermatozoa per milliliter by using conventional IVF techniques and they were added to the culture media 3 h after retrieval. Fertilization of 12 oocytes occurred 18 h after insemination (day 1). These oocytes with two pronuclei and two polar bodies were maintained for 24 h in the same culture media. Afterwards, grade 2 embryos (two at the 4‐cell stage and one at the 5‐cell stage) were transferred to the uterus by using a Phycon IVF catheter. The luteal phase was supported with 50 mg progesterone i.m. per day for 14 days.

Two weeks after the embryo transfer, her semiquantitative β‐HCG test was positive. The transvaginal ultrasound scan (21 days after retrieval) showed two gestational sacs in the uterus. At 9 weeks gestation, an ultrasound scan showed three viable fetuses in three amniotic cavities, two of which were contained in the same gestational sac. Therefore, dizygotic triplet pregnancy was diagnosed.

At 16 weeks of gestation, the patient was admitted to our hospital for the management of threatened premature delivery. The ultrasound examination at 26 weeks of gestation showed TTTS, namely, the discordant growth between the twins was shown, and the larger one showed polyhydroamnion, while the smaller one showed oligohydroamnion. The smaller fetus showed the reverse end‐diastolic velocity of umbilical artery with pulsed Doppler examination. Subsequently, intrauterine fetal death (IUFD) of the smaller fetus occurred. A cesarean section was performed at 27 weeks of gestation for premature rupture of the membranes. The birth weights were 1044 g (boy), 1040 g and 796 g of still birth (monozygotic girls). Examination of the placentae showed a dichorionic triplet pregnancy.

DISCUSSION

OUR PRESENT CASES with triplet pregnancy were composed of both dizygotic and monozygotic. It is needless to say that multiple gestations can be characterized as dizygotic or monozygotic. Twin gestation can be characterized as dizygotic or monozygotic. While dizygotic twins occur when two separate ova are fertilized by two separate sperm, monozygotic twins represent division of the fertilized ovum at various times after conception. Treatment with drugs for ovulation (clomiphene citrate and gonadotropins) is known to increase the rate of monozygotic twinning to 1.2%, which is significantly higher than that of spontaneous ovulation, 0.45%. ³ , ⁶ If division of the conceptus occurs within 3 days of fertilization, each fetus will be surrounded by an amnion and chorion, and the membranes are termed diamniotic dichorionic. If division occurs after that, the chorion has already begun to develop, whereas the amnion has not. In this case, both fetuses will be surrounded by a single chorion, a condition termed monochorionic. If division occurs between the fourth and eighth day following fertilization, each fetus will be surrounded by an amnion, and the condition is termed diamniotic monochorionic. If division occurs from day 9 to day 12, this is after development of both the amnion and the chorion, and the twins will share a common sac, a condition termed monoamniotic/monochorionic. It is reported that the incidence of monochorionicity associated with assisted reproduction is at least 3.2%, ⁷ which is higher than that in the general population. There are some possible explanations for this increased rate of monozygotic twinning in humans. The artificial conditions of culture media are the likely causes of the increased incidence of monozygotic twinning in the population of IVF patients. ⁸ Zonal manipulation was cited as a major factor in the increased incidence of monozygotic twinning ⁹ and mono amniotic twins in the IVF‐embryo transfer population. ¹⁰ Until now, there have been three reports on dizygotic triplet pregnancy and two reports on trizygotic quadruplet pregnancy in conventional IVF without micromanipulation. ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ These reports suggested that monozygosity may be independent of micromanipulation. In vitro fertilization itself is probably the cause of the increase in monozygotic pregnancy. In addition, it is reported that the zonal manipulation increases the occurrence of not only monoamniotic twins but also monozygotic twins. As the rate of zonal manipulation in IVF is increasing, the increase of monozygosity because of assisted reproductive techniques might be inevitable. High order pregnancies are associated with increased risk of maternal complications as well as a high prevalence of prenatal and neonatal morbidity and mortality. ¹⁶

Although all twins face certain risks, monozygotic twins face additional risks related to the time when twinning occurs. In case 2, one fetus died because of twin‐to‐twin transfusion syndrome (vascular anastomoses between the monozygotic fetuses). This is a result of fetofetal transfusion via placental vascular anastomoses. In this circumstance, while the donor becomes anemic and suffers growth retardation, the recipient develops polycythemia with circulatory overload and an increased urine output resulting in polyhydramnios. ¹⁷ , ¹⁸ Twin‐to‐twin transfusion syndrome typically presents in the mid trimester with polyhydramnios or oligohydramnios. The diagnosis of severe TTTS is based on the ultrasound findings of a monochorionic diamniotic twin pregnancy, with fetuses that are discordant in size. ¹⁹ Twin‐to‐twin transfusion syndrome occurs in approximately 15% of diamniotic monochorionic twin pregnancies. ²⁰ , ²¹ The survival rate with TTTS diagnosed before 28 weeks of gestation is reported to be 20–45%. ²² , ²³ Because of the increased risk of monozygosity following assisted conception procedures, a monozygotic multiple pregnancy should be carefully scanned by transvaginal sonography early in the pregnancy. Further studies of the relationship between monozygosity and in vitro conditions may contribute to the decrease of monozygotic multifetal pregnancies following IVF.