Journal of Experimental & Clinical Assisted Reproduction BioMed Central

We present a series of monozygous multiple gestations achieved following in vitro fertilization (IVF): one case of monochorionic triplet pregnancy and six cases of dizygotic triplet pregnancy. all patients achieving clinical pregnancy by ART were reviewed (n = 2433). A 37 year-old woman who delivered a healthy singleton after IVF returned two years later for FET, and a single blastocyst was transferred. This also resulted in pregnancy, but TV-USG revealed a single gestational sac with three distinct amniotic sacs, each containing a distinct fetal pole with cardiac activity. This pregnancy was electively terminated at nine weeks' gestation. An additional six cases of dizygotic triplets established after fresh embryo transfer (no ICSI or assisted hatching) are also described. Of these, one resulted in a miscarriage at eight weeks' gestation and five patients have an ongoing pregnancy. This case series suggests the incidence of dizygotic/monochorionic triplets following IVF is approximately 10 times higher than the expected rate in unassisted conceptions, and underscores the importance of a conservative approach to lower the number of embryos at transfer. The role of embryo transfer technique and in vitro culture media in the twinning process requires further study.


Background
With the advent of the advanced reproductive technologies, the rate of multiple gestation has increased. In Japan, the rate of monochorionic triplet pregnancy is reported to be 0.004% of natural pregnancies [1,2]. Although the rate of monochorionic triplet pregnancy is quite low, it has been reported that both monochorionic triplet pregnancy and monozygotic twins [3] is increased in the setting of IVF. In this report, we review features of seven unusual cases of multiple gestation established after IVF in Japan, and discuss some possible explanations for the observed findings.

Case presentation
We reviewed records of all patients who become clinically pregnant via IVF at Denentoshi Ladies Clinic Reproductive Center between September 2000 and December 2006 (n = 2,433). In all cases, embryos were uniformly transferred using a Phycon IVF Catheter (Fuji System, Tokyo, Japan) under abdominal ultrasound guidance by the same individual. Seven cases of monzygous multiple gestation were identified, as described here (Table 1):

Case 1
This Caucasian gravida 2 para 1 (age 37) presented with her partner for assessment and treatment of unexplained infertility of three years' duration. Her gynecological history was unremarkable. She had a regular 28 day menstrual cycle and all laboratory test results were normal. The couple underwent ovulation induction +IUI six times without success before opting for IVF. Following pituitary down-regulation with intranasal buserelin acetate (Buserecure, Fuji Pharmaceutical, Tokyo, Japan) initiated on day 2, controlled ovarian hyperstimulation was performed using a combined follicle stimulating hormone (FSH; Fertinom P, Serono, Tokyo, Japan) + human menopausal gonadotoropin (HMG; Humegon, Japan Organon, Osaka, Japan) protocol. Human chorionic gonadotropin 10,000 IU (hCG; Profasi, Serono, Tokyo, Japan) was administered 36 hours before retrieval. Ten oocytes were obtained via ultrasound-guided transvaginal needle aspiration, and were cultured in Universal IVF Medium (Medi-Cult a/s; Jyllinge, Denmark) and BlastAssist System 1, 2 (MediCult a/s) in a 5% CO 2 , 5% O 2 , and 90% N 2 environment. Eight oocytes were cultured for five days, and of these four developed to the blastocyst stage. The patient underwent a single blastocyst transfer (Gardner's classification: 5AA) on day 5 following ovum collection. The three non-transferred blastocysts (grade 5AA, 5AA and 4AA) were cryopreserved by vitrification (Cryotop and Vitrification Kit; Kitazato Supply, Shizuoka, Japan). Luteal progesterone support was given for two weeks. The patient achieved a pregnancy and delivered a healthy singleton infant.
Two years later, the patient returned for FET. One blastocyst (grade 5AA) was thawed using the Cryotop and the Vitrification Kit; and was transferred on day 5 with assisted hatching (AH) via acid Tyrode's method (ovulation occurred in a natural cycle). She achieved a successful pregnancy and a single gestational sac (GS) was observed at 5 weeks gestational age. Two weeks later, follow-up TV-USG confirmed a single gestational sac but three distinct amniotic sacs could now be visualized, each with distinct fetal poles and distinct cardiac activity. The risks of multiple gestation were carefully explained to the patient and the case was co-managed with a perinatologist. After extensive counseling, the couple decided to terminate the pregnancy at 9 weeks' gestation.

Six additional cases (dizygotic triplet)
Case 2 (age 32). This patient sought IVF+ICSI for male factor infertility. Pituitary down-regulation was achieved with intranasal buserelin acetate (Buserecure, Fuji Pharmaceutical, Tokyo, Japan) initiated on day 2, controlled ovarian hyperstimulation was performed using a combined follicle stimulating hormone (FSH; Fertinom P, Serono, Tokyo, Japan) + human menopausal gonadotoropin (HMG; Humegon, Japan Organon, Osaka, Japan) protocol. Human chorionic gonadotropin 10,000 IU (Profasi, Serono, Tokyo, Japan) was administered 36 hours before retrieval. The patient requested transfer of two embryos following in vitro culture for five days with Quinn's Advantage medium (SAGE In-Vitro Fertilization. Inc., CT, USA). A positive pregnancy test was confirmed 14 days post-ET, and follow-up TV-USG revealed a dichorinoic-triamniotic triplet gestation. Cesarean delivery was performed at the 33 rd gestational week without complication.
Case 3 (age 34). This patient underwent IVF+ICSI for male factor infertility using the "long protocol" controlled ovarian hyperstimulation. Following culture for five days with Quinn's Advantage medium (SAGGE In-Vitro Fertiliza- Each fetus was associated with an independent amniotic membrane, consistent with dichorinoic-triamniotic triplet. A cerclage was placed at 12 weeks' gestation. The patient was hospitalized at 33 weeks due to premature rupture of membranes, and a cesarean section was performed at 33 5/7 weeks' gestation.

Discussion
Multiple gestation is associated with well known increases in obstetrical risk; moreover, the frequency of multiple gestation increases in IVF where more than one embryo is transferred. Monochorionic triplet pregnancy represents a very rare subset of multiple gestation, and occurs only in approximately 0.004% of pregnancies [1,2]. Interestingly, some researchers have reported an incidence of monochorionic triplet pregnancy after IVF approximately 100 times higher than that occurring in natural (unassisted) pregnancy [4,5]. At our center the incidence of monochorionic triplet pregnancy was calculated at 0.048%, some 10 times higher than the rate observed in natural pregnancy. Since this ratio was calculated from clinical pregnancy data (and the successful pregnancy outcome rate was unknown), the actual ratio is likely to be higher. Although the literature contains a number of reports describing this phenomenon [6,7], the exact mechanism whereby IVF influences the frequency of monochorionic triplet pregnancy remains controversial.
Several factors have been offered to explain the mechanism whereby the advanced reproductive technologies increase the rate of monochorionic pregnancy. These include ovulation induction [8], in vitro culture conditions [9], micromanipulation of the zona pellucida [10], and patient history [11]. Assisted embryo hatching (AH) and ICSI have also been implicated as factors in the monochorionic equation [12].
Our data do not find AH and ICSI making an important contribution to the incidence of triplets, a finding consistent with data on monozygous twins in IVF reported from the Cornell group [13]. Henne et al suggested that patients who undergo AH with ICSI have an a priori poor prognosis due to advanced age [7]. Similarly, Elizur et al concluded that monozygotic twinning is not associated with zona pellucida micromanipulation procedures [14]. It has been suggested that some IVF patients may have a degree of cytoplasmic fragility and that the incidence of triplets does not increase for an independent reason other than manipulation of the zona pelucida [3]. It has been suggested that ovarian stimulation itself increases the incidence of multiple gestation. It should be noted that monochorionic triplet increases following ovulation induction, and that gonadotropin exposure during ART is one of the reasons [15]. Moreover, it is suspected that prolonged embryo culture and associated blastocyst transfer may increase the incidence of monochorionic triplet [9]. Yet many clinics now perform blastocyst transfer and it seems unlikely that this is an important reason for the increase in incidence of multiple gestation following IVF. According to one report [15], the mechanism leading to a monochorionic multiple gestation may be related to a weakening of the cell before implantation and a separation of the inner cell mass via an unknown process. The cases presented here do not shed light on this unknown mechanism. As multiple gestation increases pregnancy complications [1] and perinatal mortality [16,17], efforts to reduce such outcomes are welcome. At present, an increased risk for multiple gestation exists whenever multiple embryos are transferred with IVF. However, further study of the embryo culture microenvironment, method of ovarian stimulation process, and the approach to embryo transfer (i.e., single embryo transfer) [18] may help lower the incidence of monochorionic triplet pregnancy to the natural rate.