Abstract
Of 816 patients who became clinically pregnant by assisted reproductive techniques between September 2000 and August 2004, we experienced 10 cases (1.2%) of monozygotic twinning, and in five of these 10 cases, implantation of another embryo resulted in dizygotic triplets. Here, we report these five cases of dizygotic triplets. Fresh embryo transfer was performed in all five cases. Intracytoplasmic sperm injection or assisted hatching was not carried out in these cases. Blastocyst transfer was performed in three cases. Three embryos were transferred in case 1 (40‐year‐old female). While only two embryos were transferred in the other four cases so as to avoid triplet pregnancy, triplet pregnancies were confirmed. Triplet pregnancy was maintained in three cases, but in the other two cases, monochorionic twinning resulted in miscarriage during the first trimester. For the three patients who delivered the triplets, while the postnatal growth has been normal for all nine babies, the mothers were hospitalized for a long period of time, and an emergency cesarean section was performed on two patients. Because triplet pregnancy could not be completely prevented even when only two embryos were transferred, physicians should be sure to obtain informed consent in similar cases. (Reprod Med Biol 2005; 4: 59–64)
Keywords: dizygotic triplet pregnancy, in vitro fertilization, monozygotic twinning
INTRODUCTION
IN ASSISTED REPRODUCTIVE techniques (ART), multiple embryo transfer is commonly performed to improve the success rate of pregnancy per cycle, and the rate of multiple pregnancy following ART is high, at approximately 20% in Japan and more than 30% in the USA. While most of these multiple pregnancies are dizygotic, ART has been documented to increase the incidence of monozygotic twinning.
Compared to dizygotic twinning, monozygotic twinning has higher perinatal risks. When multiple embryos are transferred in ART, if two embryos implant and one results in monozygotic twinning, dizygotic triplet pregnancy occurs, thus further increasing the perinatal risks. We present five clinical cases of dizygotic triplet pregnancy following ART.
MATERIALS AND METHODS
WE RETROSPECTIVELY ANALYZED all clinical pregnancy conceived in our in vitro fertilization (IVF) program since September 2000, and the incidence of dizygotic triplet pregnancies following assisted reproductive techniques was examined.
Ovarian stimulation was performed using a gonadotropin‐releasing hormone agonist (buserelin acetate; Sprecure, Aventis Pharma, Tokyo, Japan) starting at the midluteal phase of the previous cycle (long protocol) or on day 2 of the cycle (short protocol), in combination with pure follicle stimulating hormone ( Fertinom P, Serono, Tokyo, Japan) or human menopausal gonadotoropin (hMG; Humegon, Japan Organon, Osaka, Japan) 150–300 IU/day. When at least two follicles had reached a diameter of >18 mm, 5000–10 000 IU human chorionic gonadotoropin (hCG; Profasi, Serono, Tokyo, Japan) was administered and oocyte retrieval was performed 35–36 h later by vaginal ultrasound‐guided follicle aspiration.
Patients underwent standard insemination or intracytoplasmic sperm injection (ICSI) as clinically appropriate, 4–6 h after oocyte retrieval. ICSI was performed if: (i) insemination by conventional IVF would have been difficult due to severe oligozoospermia or asthenozoospermia; (ii) insemination by conventional IVF had failed in the past; or (iii) fertilization rate was extremely poor.
Embryos were cultured in Quinn's Advantage medium (SAGE In‐Vitro Fertilization. Inc., CT, USA) or Universal IVF Medium and BlastAssist System 1, 2 (MediCult a/s, Jyllinge, Denmark) under a 5%CO2, 5%O2, and 90%N2 environment.
Embryos, in either the cleavage or blastocyst stage, were transferred to the uterus using a Phycon IVF Catheter (Fuji System Ltd, Tokyo, Japan) or FS‐ET Catheter (Kitazato Supply Co. Ltd, Shizuoka, Japan) under ultrasound guidance. A maximum of three embryos were transferred.
When undergoing IVF for the first time, as a general rule, embryos in the cleavage stage at 2 or 3 days after collection were transferred, but blastocyst transfer was performed if cleavage‐stage embryo transfer had repeatedly failed in the past or if the patient requested the use of less than two embryos to avoid triplet pregnancy. Cleavage‐stage embryos and blastocysts were cryopreserved by vitrification (Cryotop and Vitrification kit, Kitazato Supply Co. Ltd, Shizuoka, Japan), and additonal embryos were not transferred.
Among patients who had undergone several previous unsuccessful transfers of high‐quality embryos, assisted hatching was performed upon consent, especially when performing frozen embryo transfer.
Luteal support consisted of 50 mg of progesterone in oil (Progeston injection; Fujipharmaceutical Co. Ltd, Tokyo, Japan) initiated 2 days after oocyte retrieval and continued until the day of pregnancy testing.
A clinical pregnancy was defined as the presence of a gestational sac by transvaginal ultrasound at 5–6 weeks’ gestation.
RESULTS
BETWEEN SEPTEMBER 2000 and August 2004, 816 patients (mean age 34.1 years) became clinically pregnant by ART at our clinic. Of these 816 patients, pregnancy was achieved by conventional IVF in 460 patients, by ICSI in 318 patients, and by combination of the two methods in 38 patients. Fresh embryo transfer was performed in 614 patients, while frozen embryo transfer was performed in 202 patients. Furthermore, cleavage‐stage embryo transfer (mean number 2.10) was performed in 374 patients, blastocyst transfer (mean number 1.84) in 409 patients, and two‐step embryo transfer (mean number 2.18) in 33 patients. Of the 816 patients, assisted hatching was performed in 14 patients. Among these 816 patients, were 10 cases (1.2%) of monozygotic twins, and in five of the 10 cases, another embryo was implanted, resulting in a dizygotic triplet pregnancy. The subjects of the present study were these five cases of dizygotic triplets (Table 1).
Table 1.
Five cases of dizygotic triplets
| Case | Age (years) | Duration of infertility (years) | Past pregnancy | Past treatment | Indication for ART |
|---|---|---|---|---|---|
| 1 | 40 | 7 | 1G 0P | IVF: 5 times at another institution | Tubal factor |
| 2 | 32 | 3 | 0G 0P | IUI: 9 times at another institution | Male factor |
| 3 | 34 | 9 | 1G 0P | IUI: 6 times | Male factor |
| 4 | 32 | 3 | 0G 0P | IUI: 6 times | Anti‐sperm antibody positive |
| 5 | 30 | 2 | 0G 0P | IUI: 3 times | Male and tubal factors |
ART, assisted reproductive technique; G, gravida; IUI, intrauterine insemination; IVF, in vitro fertilization; P, para.
Table 2 shows the details of ART for the five cases. All five patients underwent conventional IVF using fresh embryos. None underwent ICSI or assisted hatching (AHA). In cases 1 and 3, eight‐cell embryos were transferred on day 3, and in the remaining three cases, blastocysts were transferred on day 5.
Table 2.
Details of the assisted reproductive techniques applied to the five patients who experienced dizygotic triplet pregnancy
| Case | Ovulation Induction | No. oocytes collected | IVF/ICSI | Day of transfer | No. embryos transferred | Quality of embryos |
|---|---|---|---|---|---|---|
| 1 | GnRH − a short hMG | 3 | IVF | D3 | 3 | 8‐cell G1, 8‐cell G2 × 2 |
| 2 | GnRH – a short hMG | 5 | IVF | D5 | 2 | 4AA, 4AB |
| 3 | GnRH – a long hMG | 12 | IVF | D5 | 2 | 3AA, 2AB |
| 4 | GnRH – a long hMG | 11 | IVF | D3 | 2 | 8‐cell G1 × 2 |
| 5 | GnRH – a long hMG | 12 | IVF | D5 | 2 | 4AA, 3AA |
Quality of embryos was assessed by Veeck's classification system on day 3 and Gardner's classification system on day 5. hMG, human menopausal gonadotoropin; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization.
Case 1 was a 40‐year‐old woman who had undergone IVF five times at another institution. At our clinic, the patient underwent IVF embryo transfer (IVFET) for the first time, and because the patient was older and had undergone IVF several times, three embryos were transferred.
The other four patients underwent intrauterine insemination three to nine times at our clinic or another clinic, and underwent IVFET for the first time at our clinic. Because these four patients were young and embryo quality was favorable, two embryos were transferred in order to avoid triplet pregnancy. The quality of embryos, as assessed by Veeck's classification system on day 3 and Gardner's classification system on day 5, was favorable or relatively favorable in all five cases.
Figure 1 shows a transvaginal ultrasound image taken on day 0 of week 8 of gestation for case 3. Two gestational sacs are visualized in the uterus, and two fetuses were seen in one of the sacs. In addition, each fetus was enclosed in an independent amniotic membrane, suggesting monochorionic diamnionic twinning.
Figure 1.
Transvaginal ultrasound image taken on day 0 of week 8 of gestation for case 3.
Table 3 shows the results of delivery. Of the five cases, two (cases 4 and 5) resulted in spontaneous abortion of the monozygotic twins during week 8 or 9 of gestation, but the singleton pregnancies were carried to full‐term. Following vaginal delivery, no abnormalities were noted in either infant.
Table 3.
Outcome of delivery, diagnosis based on the fetal membrane, and prognosis of the infants from the 5 patients with dizygotic triplet pregnancy
| Case | No. weeks at delivery (weeks/days) | Manner of Delivery | Birthweight (g) | Gender | Apgar score | External anomalies | Membranous diagnosis of monozygotic twins | Postnatal growth |
|---|---|---|---|---|---|---|---|---|
| 1 | 33/5 | Cesarean section | 2050 | ? | 9→10 | None | Monochorionic diamnionic | Normal |
| 1738 | ? | 9→10 | ||||||
| 1614 | ? | 9→10 | ||||||
| 2 | 33/4 | Cesarean section | 1830 | / | 6→8 | None | Monochorionic diamnionic | Normal |
| 1794 | ? | 8→9 | ||||||
| 1704 | ? | 7→9 | ||||||
| 3 | 30/5 | Cesarean section | 1366 | / | 5→8 | None | Monochorionic diamnionic | Normal |
| 888 | ? | 8→9 | ||||||
| 838 | ? | 6→8 | ||||||
| 4 | 37/2 | Vaginal delivery | 2765 | ? | 9→10 | None | Monochorionic diamnionic | Normal |
| 5 | 40/3 | Vaginal delivery | 2636 | / | 9→10 | None | Monochorionic diamnionic | Normal |
Underline indicates monozygotic twins in each case.
The other three patients (cases 1–3) carried their triplet pregnancies, and in all three cases, cervical cerclage was performed during 12–17 weeks of gestation.
Case 1 was hospitalized on day 0 of week 20 of gestation due to threatened premature delivery, and drip infusion of ritodrine hydrochloride was initiated. With time, leg swelling worsened, but proteinuria and hypertension were mild. The patient was continuously hospitalized for more than 3 months, and a cesarean section was performed on day 5 in week 33.
In case 2, oral administration of ritodrine hydrochloride was initiated at week 16 and the patient rested at home. The patient was hospitalized on day 6 of week 28. Leg swelling and proteinuria were mild, and there was no hypertension. While the patient was scheduled to undergo a cesarean section in week 34, an emergency cesarean section was performed on day 4 of week 33 due to premature rupture of the membrane.
Case 3 was hospitalized on day 4 of week 26 due to high rupture of the membranes, and drip infusion of ritodrine hydrochloride and antibiotics were initiated. In addition, a steroidal agent was administered to facilitate fetal lung maturation. Leg swelling and proteinuria were mild, and there was no hypertension. An emergency cesarean section was performed on day 5 of week 30 because cardiac deceleration in one of the twins was confirmed by fetal heart monitoring.
Table 3 shows the birth weights. Intrauterine growth retardation (IUGR) was confirmed in one set of monozygotic twins (case 3) and not in the other fetuses, or in any of the infants for cases 1 and 2. Also, discordancy was not seen in any of the three sets of monozygotic twins.
While some infants had low 1‐min Apgar scores, all had normal scores at 5 min. None of the nine infants showed external abnormalities, and postnatal development in all has been favorable.
Transvaginal ultrasound in early gestation and histopathological analyzes of the fetal accessory organs after delivery suggested that all five patients had monochorionic‐diamnionic twinning.
DISCUSSION
THE INCIDENCE OF monozygotic twinning in natural pregnancy has been reported at approximately 0.45%. 1 , 2 According to the literature, the incidence of monozygotic twinning following ART is higher, at approximately 1–3%. 3 , 4 , 5 , 6 , 7 , 8 At our clinic, the incidence of monozygotic twinning following ART is 1.2%, and thus is in agreement with these reports. The incidence of monozygotic twinning has been reported to be increased by a number of factors, including the use of an ovulation‐inducing agent 1 artificial conditions of in‐vitro media 3 and zona manipulation, such as ICSI or AHA. 8 , 9 , 10 However, there have been contradictory reports 6 including one hypothesis that monozygotic twinning involves apoptosis of inner cell mass cells due to in vitro culturing. 11
Several studies have found that blastocyst transfer appears to increase the incidence of monozygotic twinning. 12 , 13 Of the five cases of dizygotic triplets in the present study, blastocysts were transferred in three cases. However, of the 816 patients who became pregnant by ART, blastocysts were transferred in 409 patients, suggesting that blastocyst transfer does not necessarily increase the incidence of dizygotic triplets. Also, none of the five patients underwent ICSI or assisted hatching, and in all five patients, fresh embryos were transferred by conventional IVF. Therefore, in the present five patients, the factor(s) that increases the onset of dizygotic triplets following ART could not be clarified.
In multiple embryo transfer, if one embryo results in monozygotic twinning and another embryo is also implanted, then a dizygotic triplet pregnancy or higher‐order multiple pregnancy occurs. 14 , 15 , 16 , 17 , 18 , 19 Alikani et al. reported that another embryo was also implanted in 63% of cases of monozygotic twinning. 7 At our clinic, of the 10 cases of monozygotic twinning following ART, another embryo was also implanted in five cases, producing dizygotic triplets.
One recent study reported that the average duration of triplet pregnancy was 32.2 weeks, and the average birthweight was 1698 g. 20 When compared to singleton pregnancy, the relative risk of infant death in multiple pregnancy was fivefold for twin pregnancy and 12‐fold for triplet pregnancy. 21 According to the Japanese Vital Statistics, when compared to singleton pregnancy, the perinatal mortality rate for triplet pregnancy was 11.1‐fold higher in 1980, but decreased to 6.9‐fold higher in 1998 22 due to recent advances in perinatal management. Despite this decrease, however, the rate remains high. More than nine of 10 triplet births are preterm (less than 37 weeks of gestation), while only less than one of 10 singleton births are preterm. The average birthweight of triplets (1698 g) is approximately half that of singletons (3358 g), and the infant mortality rate for triplet pregnancy and other higher‐order multiple pregnancies is 12 times higher than that for singleton pregnancy. 23
In triplet pregnancy, the incidence of premature birth is approximately 90%, the incidence of low birthweight is more than 90%, and the incidence of very low birthweight is more than 30%. 24 In addition, when compared to singleton pregnancy, the incidence of disability is higher. 25 Monozygotic twinning is associated with a high rate of abortion and twin to twin transfusion syndrome, and since the incidence of discordancy is higher, the risks associated with monozygotic twinning are much greater than those associated with dizygotic twinning. In twinning generally, the degree of fetal discordancy is greater for higher‐order multiple pregnancy 26 and fetal and neonatal weight discordancy leads to an increased incidence of intrauterine fetal death. 27 Therefore, dizygotic triplet pregnancy has risks associated not only with triplet pregnancy, but also with monozygotic twinning.
As to the risks for mothers associated with multiple births, particularly in cases of triplets and higher‐order multiple pregnancy, the incidence of preeclampsia is 21% and that of preterm/premature rupture of the membranes is 20%. 28 Careful perinatal management is therefore needed, and in many cases, the duration of inpatient care during multiple pregnancy tends to be longer. 29 Among our five patients, three carried their pregnancies, and were hospitalized for 4–15 weeks, starting from the second trimester until delivery. While IUGR was confirmed in one set of monozygotic twins, the postnatal status for all nine infants was favorable, and all are continuing to do well. One recent study found that, due to improved obstetric management, excellent survival rates with very low associated morbidity have been obtained with triplets, and that 96% of triplets survived to discharge. 30 However, in many institutions, the perinatal risks associated with triplet pregnancy are higher than those associated with singleton pregnancy. Among the present five patients with dichorionic triplet pregnancy, monochorionic twinning resulted in miscarriage in two patients. Also, for the three patients who delivered the triplets, while the postnatal growth has been normal for all nine babies, the mothers were hospitalized for a long period of time, and an emergency cesarean section was performed on two patients.
In four of our five cases of dizygotic triplet pregnancy, we transferred only two embryos so as to avoid triplet pregnancy. Therefore, physicians should be sure to obtain informed consent in similar cases even though the chance of triplet pregnancy is low when two embryos are transferred. Furthermore, because triplet pregnancy cannot be completely prevented even when only two embryos are transferred, if the patient is young and the embryos are of good quality, physicians should actively consider single embryo transfer.
ACKNOWLEDGMENTS
THE AUTHORS EXPRESS their deep appreciation to Dr Michihiro Kitagawa of the National Center for Child Health and Development, Dr Yoshihiro Inomata of Tokyo Metropolitan Municipal Otsuka Hospital, and Dr Ichiro Yasuhi of the National Hospital Organization Nagasaki of the patients with triplet pregnancy.
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