Dizygotic twin pregnancy after single embryo transfer: a case report and review of the literature

Introduction

Elective single embryo transfer (SET) is a reasonable option for avoiding multiple pregnancies in patients who undergo assisted reproductive technology (ART) treatment [1, 2]. The Japan Society of Obstetrics and Gynecology (JSOG) recommends SET for patients under 35 years of age and for those who have undergone ≤2 treatment cycles since 2008. The JSOG recommendation increased the use of the SET technique from 49.9 % (52,800/105,812) of all embryo transfer cycles in 2007 to 73 % (106,758/146,286) in 2010 [3, 4]. As a result, the proportion of all embryo transfer cycles that resulted in multiple pregnancies decreased from 3.0 % in 2007 to 1.3 % in 2010 [3, 4].

Multiple pregnancies may occur even if SET is performed. To date, 17 cases involving twin or triplet pregnancies after the SET have been reported [5]. In the general population, monozygotic twins have been calculated to account for 0.4–0.45 % of all live births [6, 7]. In patients undergoing ART, this rate is approximately twice that of spontaneous pregnancies [8, 9]. Theoretically, twins resulting from a SET procedure should be monozygotic. However, a review of the literature suggests that 4 cases of dizygotic twin pregnancies have been reported in patients undergoing the SET technique [10–12]. Here, we present a fifth case and a review of the literature concerning this phenomenon.

Case report

A 39-year-old, nulligravida woman and her 44-year-old husband had an 8-year history of infertility. She had regular menstrual cycles occurring every 28 days and lasting 7 days, and was negative for Chlamydia trachomatis IgA and IgG antibodies. She had bilateral tubal patency by hysterosalpingography. Her husband’s semen analysis showed normozoospermia, based on the 2010 World Health Organization criteria.

The woman underwent in vitro fertilization (IVF) and embryo transfer with a long-protocol, gonadotropin-releasing hormone agonist because of unexplained infertility. Nine cumulus oocyte complexes (COCs) were aspirated and these COCs were subjected to IVF. Six fertilized oocytes with two pronuclei were observed 20 h after insemination. Although one embryo at the morula stage was transferred to the uterus on day four after IVF, she did not become pregnant. The remaining four morula-stage embryos with good morphology were subjected to vitrification in a solution containing 40 % (vol/vol) ethylene glycol, 18 % (wt/vol) Ficoll 70, and 0.3 mol/L trehalose [13]. After 2 months of IVF treatment, the patient became pregnant spontaneously, but this resulted in a biochemical pregnancy. Four months after vitrification of the embryos, four embryos were warmed for a scheduled embryo transfer, according to the patient’s natural menstrual cycle. Ovulation was confirmed by follicular monitoring with transvaginal ultrasonography on day 17 of the cycle. The embryo transfer was planned 5 days after the ovulation.

Warming of morula-stage embryos was performed on the day before the scheduled embryo transfer. Three surviving morula-stage embryos were cultured, after warming in Global Medium (LifeGlobal, Gilford, CT, USA) supplemented with 10 % synthetic serum substitute (Irvine Scientific, Santa Ana, CA, USA) for 18 h. One embryo showed further development and reached the expanded blastocyst stage. The expanded blastocyst was transferred into the patient’s uterus. Ten days after embryo transfer, her pregnancy test was positive. Two gestational sacs with fetal heart beats in each gestational sac were observed by transvaginal ultrasound in the uterus at 6 weeks’ gestation. Because a single embryo was transferred, we believed the pregnancy to be monozygotic dichorionic twins. At 37 + 0 weeks’ gestation, she delivered a 1,986 g male infant and a 2,490 g female infant by elective cesarean section. After a G-banding chromosome analysis in both infants, the phenotypically male and female infants showed 46,XY and 46,XX, respectively. Therefore, the infants were considered dizygotic twins.

Literature review

PubMed and Google Scholar literature searches of the English language publications from 1978 to 2013 were performed using the following search terms: single embryo transfer, dizygotic twins, fertilization in vitro, and sex-discordant. We found four cases of dizygotic twins after SET reported in three papers published between 2009 and 2011 [10–12]. The four cases from the literature search and our case were carefully reviewed.

Table 1 summarizes the five cases of dizygotic twins born after SET. There were two patients with tubal infertility and two patients with unexplained infertility. The cause of infertility was not documented in one case. In all cases, the husband’s semen analysis was normal. There were two cases of fresh and three cases of frozen-thawed or vitrified-warmed embryo transfer cycles. The embryo transfers were performed during a spontaneous menstrual cycle in the three patients undergoing frozen-thawed or vitrified-warmed embryo transfer cycles. Although an early cleavage-stage embryo was transferred in one patient, late-stage embryos (morula or blastocyst) were transferred in four patients. All sets of delivered twins included infants of both sexes, with a median birth weight of 2,012 g (range, 704–2,877 g). In three cases, a karyotype analysis was performed, showing normal male and female karyotypes. The placentas after delivery were micro- or macroscopically observed to confirm that they were separate in four patients. Unprotected intercourse was confirmed around the time of the embryo transfer cycles in four cases.

Table 1.

Summary of reports of dizygotic twin pregnancy after single embryo transfer

	Case 1	Case 2	Case 3	Case 4	Case 5
First author	Kyono [10]	Sugawara [11]	Sugawara [11]	van der Hoorn [12]	Takehara (present case)
Age (years)	27	35	29	30	39
Gravida	1 or more	2 or more	1 or more	0	0
Parity	1 or more	0	Not documented	0	0
Duration of infertility (years)	Not documented	9	1	Not documented	8
Cause of infertility	Not documented	Unexplained infertility	Tubal infertility	Tubal infertility	Unexplained infertility
Husband’s semen analysis	Normal	Normal	Normal	Normal	Normal
Embryo transfer cycle	Fresh embryo transfer cycle	Fresh embryo transfer cycle	Vitrified-warmed embryo transfer cycle	Frozen-thawed embryo transfer cycle	Vitrified-warmed embryo transfer cycle
Methods of ovarian stimulation	GnRH agonist-long protocol	GnRH antagonist protocol
Methods of fertilization	ICSI	IVF	ICSI	IVF	IVF
Methods of endometrium preparation in frozen-thaw cycles			Spontaneous cycle	Spontaneous cycle	Spontaneous cycle
Stages of embryos at embryo transfer	Morula stage	Blastocyst stage	Blastocyst stage	10-cell stage	Blastocyst stage
Gestational weeks at diagnosis of dichorionic twins	8 weeks	7 weeks	5 weeks	7 weeks	7 weeks
Gestational weeks at diagnosis of dizygotic twins	20 weeks	Not documented	Not documented	20 weeks	Not detected during pregnancy
Gestational weeks at delivery	37 weeks	26 weeks	36 weeks	36 weeks	37 weeks
Complications of the mother during pregnancy	Not documented	Pregnancy-induced hypertension	Not documented	Preeclampsia	None
Complications of the infants during pregnancy	Not documented	Intrauterine growth restriction	Not documented	Intrauterine growth restriction	None
Infants	1,998 g, female/2,096 g, male	704 g, female/420 g, male	2,877 g, female with neural tube defect/2,544 g, male	2,025 g, female/1,475 g, male	2,490 g female/1,986 g, male
Karyotypes of infants	46,XX/46,XY	Not tested/46,XY	Not tested/not tested	Not tested/not tested	46,XX/46,XY
Placental findings	Not documented	Two separated placentas	Two separated placentas	Two separated placentas	Two separated placentas
Other evidence of dizygotic twins	Not documented	Not documented	Not documented	Different HLA typing of the two infants	Not examined
Intercourse during the transfer cycles	Yes	Yes	Unknown	Yes	Yes

ICSI intracytoplasmic sperm injection, IVF in vitro fertilization, HLA human leukocyte antigen

Discussion

In the present study, we reported a case of dizygotic twins following SET and reviewed the literature. All five literature-reported cases of dizygotic twins were considered to have occurred as the result of SET and concurrent natural pregnancies.

Although, in general, twin pregnancies following SET are considered to be monozygotic, the five reported cases, including the present one, were diagnosed as dizygotic twins following SET because of the different infant sexes. It is difficult to determine the zygosity in twins of the same sexes. Apart from sex determination, there are several methods for determining zygosity, such as chorionicity and amnionicity by placenta and membrane findings, infant blood type, HLA typing, and DNA fingerprinting [14]. Of these methods, placental and membrane findings after delivery are simple and are extensively used to determine zygosity. In the present study, we found that separate placentas, which indicate dichorionic and diamniotic twins, developed in four of five cases. However, dichorionic twins are not necessarily dizygotic twins [15]. In contrast, monochorionic twins are widely accepted to be monozygotic [14]. In the present study, the dichorionic twins were diagnosed by ultrasonography within 8 weeks’ gestation in all cases, including the present case. However, only two cases were prenatally diagnosed as dizygotic twins due to their being different sexes. Physicians may assume that twins arising from a SET pregnancy are monozygotic; however, they should, nevertheless, carefully check chorionicity, amnionicity, and infant sex by ultrasonography even after SET.

All five case of dizygotic twins described in this paper were considered to have occurred as a result of pregnancies following SET and concurrent natural conception. In the five described instances of the conception of dizygotic twins following SET, the husband’s semen characteristics were normal and unprotected intercourse was confirmed around the time of embryo transfer in four of five cases. There is a possibility that spontaneous ovulation of the remaining oocytes after aspiration of follicles occurred during the two fresh embryo transfer cycles. Moreover, the frozen-thawed or vitrified-warmed embryo transfer cycles were performed during a spontaneous menstrual cycle, instead of during a hormone replacement cycle. We never know the actual percentage of natural pregnancies that occur during ART cycles. If the twin infants are of the same sex, after the transfer of one embryo, the percentage of monozygotic twins could be overestimated because one of the twins might have been naturally conceived [12]. Taken together, the prevalence of dizygotic twins after SET may be underestimated as zygosity may not be fully investigated in infants of the same sex.

Many IVF programs advise 2–7 days of abstinence before oocyte retrieval to maximize the semen quality and quantity. Although these guidelines certainly reduced the chance of natural conception during the IVF cycles, they do not completely prevent it [16]. Mains et al. recommend patients to abstain from vaginal intercourse starting on day six of ovarian stimulation, which is 2 days before the earliest possible day of human chorionic gonadtropin tiggering [16]. However, advice against intercourse around the time of embryo transfer is not typically given because it might have a positive effect on the pregnancy outcome. Only two studies have investigated the effects of vaginal intercourse around the time of embryo transfer during ART cycles [17, 18]. Tremellen et al. reported that, in a multicenter randomized study, patients undergoing fresh (400 cycles) and frozen-thawed (200 cycles) embryo transfer were randomized either to abstain or to engage in vaginal intercourse around the time of embryo transfer. Although there are no significant differences in the pregnancy rates between the intercourse and abstain groups, the proportion of transferred embryos that remained viable at 6–8 weeks’ gestation is significantly higher in the intercourse groups compared to the abstinence group [17]. In another study, the vaginal intercourse around the time of embryo transfer did not show positive effects on pregnancy and implantaion rates in ART cycles [18].

Because SET is recommended to prevent multiple pregnancies, we suggest that couples who undergo ART abstain from vaginal intercourse around the time of embryo transfer period, and that both patients and physicians should consider that natural pregnancy might occur during the ART cycle.