Triplet Pregnancy in a Cynomolgus Monkey (Macaca fascicularis) after Double Embryo Transfer

Abstract

At our research center, cynomolgus monkeys (Macaca fascicularis) are bred by mating or intracytoplasmic sperm injection (ICSI) and embryo transfer. We typically transfer 2 embryos, because the pregnancy rate is better than that for single embryo transfer. In the case we present here, 2 embryos that had been frozen and thawed after ICSI were transplanted into a recipient female macaque, and a multiple pregnancy (3 fetuses) was confirmed. All 3 fetuses were miscarried between days 81 and 85 of pregnancy. One fetus, which was wrapped in the amnion, was expelled along with its own placenta and one other. Because the other placenta had 2 umbilical arteries, 2 fetuses may have shared it. Therefore, we believe this pregnancy was a case of triplets, including a set of twins from an embryo that divided after transfer.

The birth of twins to rhesus² and Japanese macaque⁷ monkeys has been reported, but the probability of twins is extremely low (0.027% to 0.21%). At our institution, we carry out indoor artificial breeding of cynomolgus monkeys (Macaca fascicularis) by using a variety of techniques, including ovarian stimulation, oocyte collection, intracytoplasmic sperm injection (ICSI), and embryo transfer. Double embryo transfer typically is performed, because the pregnancy rate is better than that for single embryo transfer.

Here we describe a case of triplets generated from double embryo transfer. We believe that 1 of the 2 transplanted embryos divided after transfer.

Case Report

Experimental procedures were approved by the animal care and use committee of Shiga University of Medical Science (Shiga, Japan). Oocytes were collected from one sexually mature female cynomolgus monkey (age, 6 y; weight, 2.3 kg), and another (age, 5 y; weight, 2.5 kg) was the recipient of embryo transfer. The animal rooms were maintained at 25 ± 2 °C, with a relative humidity of 50% ± 5% and 12:12-h light:dark cycle (lights on, 0800 to 2000). Cynomolgus monkeys were housed individually (cage dimensions, 500 mm × 800 mm × 800 mm). In the morning, each animal was fed commercial pellet monkey chow (20 g per kg of body weight; CMK1, CLEA Japan, Tokyo), supplemented with 20 to 50 g of sweet potato in the afternoon. Water was given ad libitum by an automatic supplier.

Oocytes were collected by laparoscopy (LA6500, Machida Endoscope, Tokyo, Japan) from adult female monkeys that had been treated with gonadotropin.⁶ Insemination was performed by ICSI with oocytes and thawed sperm, and embryos were cultured in vitro.⁸ Three days after fertilization, 12-cell and 8-cell embryos were vitrified (Cryotop and Vitrification Kit; Kitazato BioPharma, Shizuoka, Japan).¹⁰ On day 547 after freezing, the embryos were thawed, and the 8-cell and 12-cell embryos were cultured for 16 h. The 8-cell embryo was confirmed to have a normal form (that is, showing neither death nor degeneration of the blastomere), and the 12-cell embryo had developed to the 16- to 32-cell stage (Figure 1). Both embryos were transplanted by laparoscopy through the fimbria into the oviduct of the recipient female macaque^8,9 at 0 to 4 d after ovulation. Pregnancy was determined by ultrasonography (SSD-620, Aloka, Tokyo, Japan) 37 d after embryo transfer. The presence of at least 2 heartbeats confirmed a multiple pregnancy (Figure 2).

Figure 1.

Cynomolgus monkey embryos (A, D) before freezing, (B, E) after thawing, and (C, F) at transfer. (A through C) Embryo that was frozen–thawed at 12 cells and transferred to the recipient at 16 to 32 cells after 16 h of culture. (D through F) Embryo frozen–thawed at 8 cells and transferred to the recipient at 8 cells after 16 h of culture. Bar, 50 µm.

Figure 2.

Ultrasonographic image of the pregnancy on day 37.

At our research center, the number of days (mean ± 1 SD; n = 14) from ICSI to birth is 157 ± 8. The first miscarriage (fetus A; Figure 3) in the macaque we present here occurred on day 81 after ICSI (not counting the freezing time). Ultrasonography after miscarriage confirmed the presence of a heartbeat. Two days later (day 83), a second miscarriage (fetus B; Figure 3) occurred, with the third miscarriage (fetus C) on day 85. Fetus C was expelled with its placenta. Another placenta was expelled on the same day. Because this second placenta had 2 umbilical arteries (Figure 3), we believe that fetuses A and B had shared it, suggesting that they were monozygotic twins; fetus C, which had a separate placenta, likely originated from the second embryo. However, because we did not perform DNA analysis, we cannot exclude the possibility that the 3 fetuses were monozygotic triplets.

Figure 3.

Dissection view of 3 miscarried fetuses. (A) Fetus (fetus A; male; length, 14 cm; weight, 35.0 g) miscarried on day 81 of pregnancy (25 January 2010). The tips of both feet were lost. The positions and shapes of the internal organs were normal, and the complexion was poor. (B) Fetus (fetus B; male; length, 15 cm; weight, 30.1 g) miscarried on day 83 of pregnancy (27 January 2010). Autolysis was pronounced, and portions of various internal organs had been resorbed. (C) Fetus (fetus C; male; length, 15 cm; weight, 51.1 g) miscarried on day 85 of pregnancy (29 January 2010). Fetus C was expelled while still wrapped in the amnion. The positions and shapes of the internal organs were normal, and it had its own placenta (weight, 19.2 g). Development of the internal organs was more advanced than in fetus A. (D) Appearance of fetus C when it was found. (E) Placenta (weight, 37.4 g) that was expelled with that of fetus C. We believe that fetuses A and B shared this placenta, because it had 2 umbilical arteries (arrows).

At our research center, double embryo transfer of cynomolgus macaques is performed to improve the pregnancy rate, and twin pregnancies do occur. Typically, however, one or both of the twins are miscarried. In addition, multiple pregnancies from single embryo transfer had not occurred previously at our institution, nor had triplets resulted from double embryo transfer. In the current case, natural separation after the 8-cell stage led to monozygotic multiple fetuses; to the best of our knowledge, we are the first to report such a result after assisted reproductive methods in cynomolgus monkeys. Embryo splitting similarly led to a twin pregnancy in a rhesus monkey, but one twin was miscarried.⁵ Although the 2 heartbeats confirmed on day 37 led us to diagnose a twin pregnancy, we now believe that it actually was a triple pregnancy. We were unable to perform chromosomal and DNA analysis on any of the 3 fetuses described herein.

Monozygotic multiples and dizygotic quadruplets and quintuplets are well known to occur in humans.^3,4 The incidence of human monozygotic twins after assisted reproductive technologies varies depending on the number of days in embryo culture and the fertilization method, such as in vitro fertilization and ICSI. Ovary stimulation and expanded blastocyst transfer are other possible causes of monozygotic twins in humans.¹

At our research center, we have encountered many twin pregnancies after double embryo transfer. However, except for 3 offspring raised by use of artificial nursing, both twins have never survived infancy. Maintaining multiple pregnancies is difficult in macaques, because they generally give birth to only one fetus.^2,7 Methods for maintaining multiple pregnancies in and managing the birth process of cynomolgus monkeys need to be improved. The current case is an unusual example of double embryo transfer leading to triplet pregnancy in cynomolgus monkeys.